Instruments, modules, and methods for improved detection of edited sequences in live cells

ABSTRACT

The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/844,330, filed 9Apr. 2020; which is a continuation of U.S. Ser. No. 16/687,640, filed 18Nov. 2019, now U.S. Pat. No. 10,625,212; which is a continuation of U.S.Ser. No. 16/540,606, filed 14 Aug. 2019, now U.S. Pat. No. 10,532,324;which claims priority to U.S. Provisional Application Nos. 62/718,449,filed 14 Aug. 2018; 62/735,365, filed 24 Sep. 2018; 62/781,112, filed 18Dec. 2018; 62/779,119, filed 13 Dec. 2018; 62/841,213, filed 30 Apr.2019. This application is also a continuation U.S. Ser. No. 16/820,292,filed 16 Mar. 2020; which is a continuation of U.S. Ser. No. 16/597,826,filed 9 Oct. 2019, now U.S. Pat. No. 10,633,626; which is a continuationof U.S. Ser. No. 16/454,865, filed 27 Jun. 2019, now U.S. Pat. No.10,550,363; which is a continuation of U.S. Ser. No. 16/399,988, filed30 Apr. 2019, now U.S. Pat. No. 10,533,152.

FIELD OF THE INVENTION

This invention relates to improved instruments, modules, and methods toscreen, select and thus optimize detection of genome edits in livecells.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that themethods referenced herein do not constitute prior art under theapplicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowfor manipulation of gene sequences, and hence gene function. Thenucleases include nucleic acid-guided nucleases, which enableresearchers to generate permanent edits in live cells. Current protocolsemploying nucleic acid-guided nuclease systems typically utilizeconstitutively-expressed nuclease components to drive high efficiencyediting. However, in pooled or multiplex formats constitutive expressionof editing components can lead to rapid depletion of edited cell typesand selective enrichment of cells that have not been edited. This occursin most cell types because only a small fraction (<1-5%) of cellssurvive the introduction of double-strand DNA (dsDNA) breaks and thusthese cells contribute fewer numbers to viable cell counts in theresulting populations compared to unedited cells that did not experiencea dsDNA break.

There is thus a need in the art of nucleic acid-guided nuclease geneediting for improved instruments, modules and methods for creatinggenome edits and for identifying and enriching cells that have beenedited. The present invention satisfies this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides instruments, modules and methods toenable automated high-throughput and extremely sensitive screening toidentify edited cells in populations of cells that have been subjectedto nucleic acid-guided nuclease editing. The instruments, modules, andmethods take advantage of isolation or substantial isolation, where theterm “isolation” in this context refers to the process of separatingcells and growing them into clonally-isolated formats. The term“substantial isolation” refers to the process of separating cells in apopulation of cells into “groups” of 2 to 100, or 2 to 50, andpreferably 2 to 10 cells. Isolation (or substantial isolation), followedby an initial period of growth (e.g., incubation), editing, and growthnormalization leads to enrichment of edited cells. Further, theinstruments, modules, and methods described herein facilitate “cherrypicking” of edited cell colonies, allowing for direct selection ofedited cells.

Isolation or substantial isolation assists in overcoming the growth biasfrom unedited cells that occurs under competitive growth regimes such asin bulk liquid culture. Indeed, it has been determined that removinggrowth rate bias via isolation or substantial isolation, incubation,editing and normalization improves the observed editing efficiency by upto 4× (from, e.g., 10% to 40% absolute efficiency at population scale)or more over conventional methods, and further that cherry-pickingcolonies using the methods described herein brings the observed editingefficiency up to 8× (from, e.g., 10% to 80% absolute efficiency atpopulation scale) over conventional methods. In some embodiments thecompositions and methods employ inducible guide RNA (gRNA) constructsleading to increased observed transformation efficiency andautomation-friendly control over the timing and duration of the editingprocess.

One particularly facile module or device for isolation or substantialisolation is a solid wall device where cells are substantially isolated,grown in a clonal (or substantially clonal) format, edited, and eithernormalization or cherry picking is employed. The solid wall devices ormodules and the uses thereof are described in detail herein. Theinstruments, modules and methods in some embodiments allow fornormalization of edited and unedited cell colonies. Normalization refersto growing colonies of cells—whether edited or unedited—to terminalsize; that is, growing the cells until the cells in the colonies entersenescence due to, e.g., nutrient exhaustion or constrained space forfurther growth. Since unedited cells grow more quickly, unedited cellcolonies will reach terminal size (e.g., senescence) before edited cellcolonies; however, the unedited cell colonies eventually “catch up” insize and senescence. Normalization of cell colonies enriches for editedcells as edited cells get “equal billing” with unedited cells.Additionally, the instruments, modules, and methods facilitate “cherrypicking” of colonies. Cherry picking allows for direct selection ofedited cells by taking advantage of edit-induced growth delay in editedcolonies. Cherry picking can be performed by selecting slow-growing cellcolonies, or cherry picking can be performed by eliminatingfaster-growing cell colonies by, e.g., irradiating the faster-growingcell colonies. Cherry picking colonies using the instruments, modules,and methods described herein may more than double the observed editingefficiency as the result of isolation or substantial isolation alone.

Certain embodiments of the instruments, modules, and methods provide forenriching for edited cells during nucleic acid-guided nuclease editing,where the methods comprise transforming cells with one or more vectorscomprising a promoter driving expression of a nuclease, a promoterdriving transcription of a guide nucleic acid and a donor DNA sequence;diluting the transformed cells to a cell concentration sufficient tosubstantially isolate the transformed cells on a substrate; growing(e.g., incubating) the substantially isolated cells on the substrate;providing conditions for editing; and either 1) growing the cellcolonies to colonies of terminal size (e.g., normalizing the cellcolonies) and harvesting the normalized cell colonies; or 2) monitoringthe growth of cells colonies on the substrate then selectingslow-growing colonies. In some aspects at least the gRNA is optionallyunder the control of an inducible promoter.

Thus in some embodiments there is provided a singulation assembly for asolid wall isolation or substantial isolation, incubation, editing, andnormalization or cherry-picking (“solid wallinsolation/incubation/normalization module” or “SWIIN”) modulecomprising: a solid wall isolation, induction and normalization (SWIIN)module comprising: a retentate member comprising: an upper surface and alower surface and a first and second end, an upper portion of aserpentine channel defined by raised areas on the lower surface of theretentate member, wherein the upper portion of the serpentine channeltraverses the lower surface of the retentate member for about 50% toabout 90% of the length and width of the lower surface of the retentatemember; at least one port fluidically connected to the upper portion ofthe serpentine channel; and a reservoir cover at the first end of theretentate member; a permeate member disposed under the retentate membercomprising: an upper surface and a lower surface and a first and secondend, a lower portion of a serpentine channel defined by raised areas onthe upper surface of the permeate member, wherein the lower portion ofthe serpentine channel traverses the upper surface of the permeatemember for about 50% to about 90% of the length and width of the uppersurface of the permeate member, and wherein the lower portion of theserpentine channel is configured to mate with the upper portion of theserpentine channel to form a mated serpentine channel; at least one portfluidically connected to the lower portion of the serpentine channel;and a first and second reservoir at the first end of the permeatemember, wherein the first reservoir is fluidically connected to the atleast one port in the retentate member and the second reservoir isfluidically connected to the at least one port in the permeate member; aperforated member comprising at least 25,000 perforations disposed underand adjacent to the retentate member; a filter disposed disposed underand adjacent to the perforated member and above and adjacent to thepermeate member; and a gasket disposed on top of the reservoir cover ofthe retentate member, wherein the gasket comprises a reservoir accessaperture and a pneumatic access aperture for each reservoir.

In some aspects of this embodiment, the permeate member furthercomprises ultrasonic tabs disposed on the raised areas on the uppersurface of the permeate member and at the first and second end of thepermeate member, the retentate member further comprises recesses for theultrasonic tabs disposed in the raised areas on the lower surface and atthe first and second end of the retentate member, the ultrasonic tabsare configured to mate with the recesses for the ultrasonic tabs, andthe permeate member, retentate member, where the perforated member andthe filter are coupled together by ultrasonic welding. In other aspectsof this embodiment, the permeate member, retentate member, theperforated member and the filter are coupled together by solventbonding.

In some aspects, the first and second reservoirs are each fluidicallycoupled to a reservoir port into which fluids and/or cells flow from thefirst retentate reservoir into the first retentate port and from thefirst permeate reservoir into the first permeate port and into theserpentine channels in the retentate and permeate members.

In some aspects of this embodiment, the SWIIN module further comprises athird and a fourth reservoir, wherein the third reservoir is 1)fluidically coupled to a second port in the retentate member, 2)fluidically coupled to a reservoir access aperture into which fluidsand/or cells flow from outside the SWIIN module into the thirdreservoir, and 3) pneumatically coupled to a pressure source; andwherein the fourth reservoir is 1) fluidically coupled to a second portin the permeate member, 2) fluidically coupled to a reservoir accessaperture into which fluids and/or cells flow from outside the SWIINmodule into the fourth reservoir, and 3) pneumatically coupled to apressure source.

In some aspects of this embodiment, the perforated member comprises atleast 100,000 perforations, or at least 200,000 perforations, or atleast 250,000 perforations, or at least 300,000 perforations, or atleast 350,000 perforations or at least 400,000 perforations, or at least500,000 perforations or more. In some aspects, the volume of a wellformed by a perforation is from 1-15 nl, or from 2-10 nl, or from 3-8nl, or from 1-10 nl, or from 2-5 nl.

In some aspects, the retentate member is fabricated from polycarbonate,cyclic olefin co-polymer, or poly(methyl methylacrylate).

In some aspects of the SWIIN module, a serpentine channel portion ofeach of the retentate and permeate members is from 75 mm to 350 mm inlength, from 50 mm to 250 mm in width, and from 2 mm to 15 mm inthickness, and from 150 mm to 250 mm in length, from 100 mm to 150 mm inwidth, and from 4 mm to 8 mm in thickness.

In some aspects, the volume of the mated serpentine channel is from 4 to40 mL, or from 6 mL to 30 mL, or from 10 mL to 20 mL. In some aspects,the volume of the first and second reservoir is from 4 to 50 mL, or from8 to 40 mL, or from 10 to 30 mL.

In some aspects of the SWIIN module, there is a support on each end ofthe permeate member configured to elevate the permeate and retentatemembers above the at least one port in the in retentate member and theat least one port in the permeate member.

Certain embodiments of the SWIIN module further comprise imaging meansto detect cells growing in the wells, and in some aspects, the imagingmeans is a camera with means to backlight the serpentine channel portionof the SWIIN. In some aspects, the SWIIN module is part of a SWIINassembly comprising a heated cover, a heater, a fan, and athermoelectric control device.

Also provided herein is an automated multi-module cell editinginstrument comprising: a SWIIN module, a housing configured to house allof some of the modules; a receptacle configured to receive cells; one ormore receptacles configured to receive nucleic acids; a growth module; atransformation module configured to introduce the nucleic acids into thecells; and a processor configured to operate the automated multi-modulecell editing instrument based on user input and/or selection of apre-programmed script.

In some aspects of the automated multi-module cell editing instrument,the transformation module comprises a flow-through electroporationdevice; and in some aspects the automated multi-module cell editinginstrument further comprises a cell concentration module. In someaspects the cell concentration module is a tangential flow filtrationmodule. In some aspects a liquid handling system transfers liquidsbetween the modules. And in some aspects, the automated multi-modulecell processing system performs the processes of growing cells,concentrating and rendering the cells electrocompetent, transforming thecells with nucleic acid-guided nuclease editing components, isolatingthe transformed cells, inducing editing in the isolated cells, andgrowing and enriching the cells, all without human intervention.

Other embodiments provide a method for enriching edited cells duringnucleic acid-guided nuclease editing comprising: transforming cells withone or more vectors comprising a promoter driving transcription of acoding sequence for a nuclease, a promoter driving transcription of aguide nucleic acid and a DNA donor sequence; diluting the transformedcells to a cell concentration to substantially isolate the transformedcells on a substrate; growing the cells and initiating editing; growingthe edited cells into colonies; and selecting cells from thesubstantially isolated colonies from the substrate or pooling cells fromthe isolated colonies from the substrate, wherein the selected cells orpooled cells are enriched for edited cells. In optional aspects of thismethod, the gRNA is under the control of an inducible promoter and 1)the cells are allowed to grow from 2-200 doublings after isolation, and2) there is an inducing step after the growth step and prior to theediting step.

Yet other embodiments provide a method for enriching edited cells duringnucleic acid-guided nuclease editing comprising: transforming cells withone or more vectors comprising a promoter driving transcription of acoding sequence for a nuclease, a promoter driving transcription of aguide nucleic acid and a DNA donor sequence; diluting the transformedcells to a cell concentration to substantially isolate the transformedcells on a substrate; growing the cells and allowing the cells to edit;growing the cells to form colonies; and selecting small colonies fromthe substantially isolated colonies from the substrate, wherein theselected cells are enriched for edited cells. In optional aspects ofthis method, the gRNA is under the control of an inducible promoterand 1) the cells are allowed to grow from 2-200 doublings aftersingulation, and 2) there is an inducing step after the growth step andprior to the editing step.

Additionally, other embodiments provide a method for enriching editedcells during nucleic acid-guided nuclease editing comprising:transforming cells with one or more vectors comprising a promoterdriving transcription of a coding sequence for a nuclease, a promoterdriving transcription of a guide nucleic acid and a DNA donor sequence;diluting the transformed cells to a cell concentration to substantiallyisolate the transformed cells on a first substrate; growing the cellsand allowing the cells to edit; and growing the cells to form coloniesof terminal size. In some aspects, the terminal-size cell colonies arepooled, and in some aspects, the terminal-size colonies are picked. Inoptional aspects of this method, the gRNA is under the control of aninducible promoter and 1) the cells are allowed to grow from 2-200doublings after singulation, and 2) there is an inducing step after thegrowth step and prior to the editing step.

Thus in some embodiments there is provided a singulation assembly for asolid wall isolation or substantial isolation, growth, induction ofediting, and normalization or cherry-picking (“solid wallinsolation/induction/normalization module” or “SWIIN”) modulecomprising: a retentate member comprising an upper surface and a lowersurface, wherein the retentate member comprises at least one retentatedistribution channel which traverses the retentate member from its uppersurface to its lower surface and for most of the length of retentatemember; wherein the lower surface of the retentate member comprisesretentate ridges between which are retentate flow directors; wherein theretentate member further comprises one or more retentate member portsconfigured to supply cells and fluid to and remove cells and fluid fromthe retentate member; and wherein the retentate member ports arefluidically-connected to the retentate distribution channel andretentate flow directors; a perforated member with an upper surface anda lower surface, wherein the upper surface of the perforated member ispositioned beneath and adjacent to the lower surface of the retentatemember and wherein the perforated member comprises at least 25,000perforations; a filter with an upper surface and a lower surface,wherein the upper surface of the filter is positioned beneath andadjacent to the lower surface of the perforated member and wherein thelower surface of the filter is positioned above and adjacent to an uppersurface of a permeate member; a gasket surrounding the perforated memberand the filter; and the permeate member comprising the upper surface anda lower surface, wherein the permeate member comprises at least onepermeate distribution channel which traverses the permeate member fromits lower surface to its upper surface and for most of the length ofpermeate member; wherein the upper surface of the permeate membercomprises permeate ridges between which are permeate flow directors;wherein the permeate member further comprises one or more permeatemember ports configured to supply fluid to and remove fluid from thepermeate member; and wherein the permeate member ports arefluidically-connected to the permeate distribution channel and permeateflow directors; and means to couple the retentate member, perforatedmember, filter, gasket and permeate member.

In some aspects of the singulation assembly embodiment the means tocouple the retentate member, perforated member, filter, gasket andpermeate member comprises ultrasonic welding and in other aspects, themeans comprises pressure sensitive adhesive, solvent bonding, matedfittings, or a combination of adhesives, welding, solvent bonding, andmated fittings; and other such fasteners and couplings. In some aspectsof the singulation assembly, the perforated member comprises at least50,000; 100,000; 150,000; 200,000, 250,000 perforations or more, and insome aspects, the SWIIN is a compound SWIIN and each part of thecompound SWIIN comprises a perforated member with at least 50,000;100,000; 150,000; 200,000, 250,000 perforations. In some aspects, theretentate and permeate members are fabricated from polycarbonate, cyclicolefin co-polymer, or poly(methyl methylacrylate); and in some aspects,the retentate and permeate members are from 75 mm to 350 mm in length,from 50 mm to 250 mm in width, and from 2 mm to 15 mm in thickness. Insome aspects of the singulation assembly, the retentate and permeatemembers are from 150 mm to 250 mm in length, from 100 mm to 150 mm inwidth, and from 4 mm to 8 mm in thickness. In some aspects of thesingulation assembly, there are two permeate distribution channelsand/or two retentate distribution channels, and in some aspects theretentate distribution channels and or permeate distribution channelsare approximately 150 mm in length and 1 mm in width. In some aspects,the retentate and/or permeate ridges are approximately 0.5 mm in heightand 80 mm in length, and in some aspects, the retentate and/or permeateflow directors are approximately 5 mm across. In some aspects, thevolume of the singulation assembly is from 15 mL to 100 mL.

Some embodiments of the disclosure provide a SWIIN module comprising thesingulation assembly and further comprising: a reservoir assemblycomprising at least two reservoirs wherein a first reservoir is 1)fluidically-coupled to at least one reservoir port into which fluidsand/or cells flow from outside the SWIIN module into the firstreservoir, 2) fluidically-coupled to a reservoir/channel port from whichfluids and/or cells flow into the one or more retentate member ports;and 3) pneumatically-coupled to a pressure source; a second reservoiris 1) fluidically-coupled to at least one reservoir port into whichfluids flow from outside the SWIIN module into the second reservoir, 2)fluidically-coupled to a reservoir/channel port from which fluids flowinto the one or more permeate member ports; and 3) pneumatically-coupledto a pressure source; and a SWIIN cover. In some aspects of the SWIINmodule embodiment, the SWIIN module further comprises two additionalreservoirs wherein a first and third reservoir are 1)fluidically-coupled to at least two reservoir ports into which fluidsand/or cells flow from outside the SWIIN module into the first and thirdreservoirs, 2) fluidically-coupled to a reservoir/channel port fromwhich fluids and/or cells flow into the at least two retentate memberports; and 3) pneumatically-coupled to a pressure source; and a secondand fourth reservoir are 1) fluidically-coupled to at least tworeservoir ports into which fluids flow from outside the SWIIN moduleinto the second and fourth reservoirs, 2) fluidically-coupled to areservoir/channel port from which fluids flow into the at least twopermeate member ports; and 3) pneumatically-coupled to a pressuresource.

In some aspects of the SWIIN module embodiment, the SWIIN covercomprises a reservoir cover portion of the SWIIN cover, and wherein thereservoir cover portion comprises 1) at least two reservoir accessapertures, wherein the reservoir access apertures provide access to thereservoir ports, and 2) at least two pneumatic access apertures, whereinthe pneumatic access apertures provide access to the at least tworeservoirs and provide negative and positive pressure to the at leasttwo reservoirs. In some aspects the SWIIN module is configured tomonitor cell colony growth after inducing editing, and further comprisesmeans for cherry picking slow growing cell colonies. In some aspects ofthe SWIIN module, editing is induced by an inducible promoter that is atemperature inducible promoter, and temperature to induce transcriptionof the nuclease and/or guide nucleic acid is provided to the SWIINmodule by a Peltier device.

In some aspects of this embodiment, the SWIIN module is one module in anautomated multi-module cell processing instrument.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a simplified flow chart of exemplary methods for enrichingand selecting edited cells. FIG. 1B is a plot of optical density vs.time showing the growth curves for edited cells (dotted line) andunedited cells (solid line). FIG. 1C depicts an exemplary inducibleexpression system for regulating gRNA and/or nuclease transcription.

FIG. 2A depicts a prior art, standard protocol for performing nucleicacid-guided nuclease genome editing. FIGS. 2B-2F depict improvedprotocols for editing in bacterial systems employing isolation orsubstantial isolation, optional induction of editing, and eithernormalization or cherry picking (e.g., selection) for identifying editedcells in a population of cells that have undergone nucleic acid-guidednuclease genome editing.

FIG. 2B depicts a protocol for functional deconvolution of the editingprocess, either by arraying cells in 96-well plates containing differentmedia or by arraying cells on a culture dish containing different media.FIG. 2C depicts a protocol for picking colonies from a culture dish,arraying the colonies on a 96-well plate, then performing functionaldeconvolution. FIG. 2E depicts a protocol for cherry picking, and FIG.2F depicts a protocol used for confirming that cherry-picking isextremely effective for selecting for edited cells.

FIG. 3A depicts a simplified graphic of a workflow for isolating,editing and normalizing cells in a solid wall device. 3B depicts asimplified graphic of a workflow variation for substantially isolating,editing and normalizing cells in a solid wall device. FIG. 3C is aphotograph of one embodiment of a solid wall device. FIGS. 3D-3F arephotographs of E. coli cells largely isolated (via substantial Poissondistribution) and grown into colonies in microwells in a solid walldevice with a permeable bottom at low, medium, and high magnification,respectively. FIGS. 3G-3J are photographs of the perforated member andthe microwells therein.

FIG. 4A-4H depict the components of two exemplary embodiments of asingulation assembly comprising retentate and permeate members, as wellas a perforated member/filter/gasket assembly. FIG. 4I-4O depict anassembled isolation, incubation, editing and either normalization orcherry-picking module (e.g., “solid wallisolation/incubation/normalization module” or “SWIIN”) and a reservoirmember to deliver fluids to the SWIIN module. FIG. 4P is an exemplarypneumatic architecture diagram for the SWIIN module described inrelation to FIGS. 4A-4P, with the status of the components for thevarious steps listed in Tables 1 and 2. FIGS. 4Q-4X depict a differentembodiment of a SWIIN module, where the retentate and permeate membersare coincident with reservoir assembly. FIG. 4Y depicts the embodimentof the SWIIN module in FIGS. 4Q-4X further comprising a heater and aheated cover. FIG. 4Z is an exemplary pneumatic architecture diagram forthe SWIIN module described in relation to FIGS. 4Q-4X, with the statusof the components for the various steps listed in Tables 3-5. FIGS.4AA-4DD are simplified depictions of the status of pressure and volumefor each reservoir in the SWIIN depicted in relation to FIGS. 4Q-4X.

FIGS. 5A-5D depict a stand-alone, integrated, automated multi-moduleinstrument and components thereof, including an isolation module, withwhich to generate and identify edited cells.

FIG. 6A depicts one embodiment of a rotating growth vial for use with acell growth module described herein. FIG. 6B illustrates a perspectiveview of one embodiment of a rotating growth device in a cell growthmodule housing. FIG. 6C depicts a cut-away view of the cell growthmodule from FIG. 6B. FIG. 6D illustrates the cell growth module of FIG.6B coupled to LED, detector, and temperature regulating components.

FIG. 7A is a model of tangential flow filtration employed by the TFFdevice presented herein. FIG. 7B depicts a top view of a lower member ofone embodiment of an exemplary TFF device. FIG. 7C depicts a top view ofupper and lower members and a membrane of an exemplary TFF device. FIG.7D depicts a bottom view of upper and lower members and a membrane of anexemplary TFF device. FIGS. 7E-7K depict various views of yet anotherembodiment of a TFF module having fluidically coupled reservoirs. FIG.7L is an exemplary pneumatic architecture diagram for the TFF moduledescribed in relation to FIGS. 7E-7K.

FIGS. 8A and 8B are top perspective and bottom perspective views,respectively, of flow-through electroporation devices (here, there aresix such devices co-joined). FIG. 8C is a top view of one embodiment ofan exemplary flow-through electroporation device. FIG. 8D depicts a topview of a cross section of the electroporation device of FIG. 8C. FIG.8E is a side view cross section of a lower portion of theelectroporation devices of FIGS. 8C and 8D.

FIG. 9 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising anisolation or substantial isolation/incubation/editing and normalizationor cherry-picking module (“solid wall isolation/incubation/normalizationmodule” or “SWIIN”).

FIG. 10 is a simplified block diagram of an alternative embodiment of anexemplary automated multi-module cell processing instrument comprisingan isolation or substantial isolation/incubation/editing andnormalization or cherry-picking module (“solid wallisolation/incubation/normalization module” or “SWIIN”).

FIG. 11A is a map of an exemplary bacterial engine vector that may beused in the methods described herein; and FIG. 11B is a map of anexemplary bacteria editing vector (with an editing cassette) that may beused in the methods described herein.

FIG. 12 is a bar graph showing the transformation efficiencies observedfor galK gRNA targeting cassettes under a variety of promoters.

FIG. 13 is a plot of cell growth vs. time, demonstrating that thermalinduction of editing does not impact bacterial cell growth or viability.

FIG. 14 depicts results demonstrating repressed gRNA cassettes yieldhigh cell viability/transformation efficiency for three exemplarynucleases.

FIG. 15 illustrates heat maps and growth curves showing the OD at 6hours for uninduced and induced cell populations.

FIG. 16 shows the results of cell colony normalization for E. coli cellsunder various conditions.

FIG. 17A is a photograph of a solid wall device with a permeable bottomon agar, on which yeast cells have been substantially isolated and growninto clonal colonies. FIG. 17B presents photographs of yeast colonygrowth at various time points.

FIG. 18 is a graph comparing the percentage of editing obtained for astandard plating protocol (SPP), and replicate samples using twodifferent conditions in a solid wall isolation, incubation, andnormalization device (SWIIN): the first with LB+arabinose; and thesecond with SOB followed by SOB+arabinose.

FIG. 19 is a bar graph showing the curing efficiency obtained with fourdifferent protocols performed for curing on the SWIIN.

FIG. 20A shows two exemplary depletion maps for MAD7. FIG. 20B is a bargraph demonstrating that the cherry-picking methods described hereinallow for identification of edits adjacent non-canonical PAMs.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentare intended to be applicable to the additional embodiments describedherein except where expressly stated or where the feature or function isincompatible with the additional embodiments. For example, where a givenfeature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polymer array synthesis, hybridizationand ligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., eds., Genome Analysis: A Laboratory Manual Series (Vols.I-IV) (1999); Weiner, Gabriel, Stephens, eds., Genetic Variation: ALaboratory Manual (2007); Dieffenbach, Dveksler, eds., PCR Primer: ALaboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: AMolecular Cloning Manual (2003); Mount, Bioinformatics: Sequence andGenome Analysis (2004); Sambrook and Russell, Condensed Protocols fromMolecular Cloning: A Laboratory Manual (2006); Stryer, Biochemistry (4thEd.) W.H. Freeman, New York N.Y. (1995); Gait, “OligonucleotideSynthesis: A Practical Approach” (1984), IRL Press, London; Nelson andCox, Lehninger, Principles of Biochemistry 3^(rd) Ed., W. H. FreemanPub., New York, N.Y. (2000); Berg et al., Biochemistry, 5^(th) Ed., W.H.Freeman Pub., New York, N.Y. (2002); Doyle & Griffiths, eds., Cell andTissue Culture: Laboratory Procedures in Biotechnology, Doyle &Griffiths, eds., John Wiley & Sons (1998); G. Hadlaczky, ed. MammalianChromosome Engineering—Methods and Protocols, Humana Press (2011); andLanza and Klimanskaya, eds., Essential Stem Cell Methods, Academic Press(2011), all of which are herein incorporated in their entirety byreference for all purposes. CRISPR-specific techniques can be found in,e.g., Appasani and Church, Genome Editing and Engineering from TALENsand CRISPRs to Molecular Surgery (2018); and Lindgren and Charpentier,CRISPR: Methods and Protocols (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”and/or “outer” that may be used herein merely describe points ofreference and do not necessarily limit embodiments of the presentdisclosure to any particular orientation or configuration. Furthermore,terms such as “first,” “second,” “third,” etc., merely identify one of anumber of portions, components, steps, operations, functions, and/orpoints of reference as disclosed herein, and likewise do not necessarilylimit embodiments of the present disclosure to any particularconfiguration or orientation.

Additionally, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, methods and cell populations that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of ordinary skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotides thatare hydrogen bonded to one another with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds. In general, a nucleicacid includes a nucleotide sequence described as having a “percentcomplementarity” or “percent homology” to a specified second nucleotidesequence. For example, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TTAGCTGG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus by homologousrecombination using nucleic acid-guided nucleases. For homology-directedrepair, the donor DNA must have sufficient homology to the regionsflanking the “cut site” or site to be edited in the genomic targetsequence. The length of the homology arm(s) will depend on, e.g., thetype and size of the modification being made. For example, the donor DNAwill have at least one region of sequence homology (e.g., one homologyarm) to the genomic target locus. In many instances and preferably, thedonor DNA will have two regions of sequence homology (e.g., two homologyarms) to the genomic target locus. Preferably, an “insert” region or“DNA sequence modification” region—the nucleic acid modification thatone desires to be introduced into a genome target locus in a cell—willbe located between two regions of homology. The DNA sequencemodification may change one or more bases of the target genomic DNAsequence at one specific site or multiple specific sites. A change mayinclude changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 or more base pairs of the target sequence. Adeletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 ormore base pairs of the target sequence. The donor DNA optionally furtherincludes an alteration to the target sequence, e.g., a PAM mutation,that prevents binding of the nuclease at the PAM or spacer in the targetsequence after editing has taken place.

As used herein, “enrichment” refers to enriching for edited cells byisolation or substantial isolation of cells, initial growth of cellsinto cell colonies (e.g., incubation), editing (optionally induced,particularly in bacterial systems), and growing the cell colonies intoterminal-sized colonies (e.g., saturation or normalization of colonygrowth). As used herein, “cherry picking” or “selection of edited cells”refers to the process of using a combination of isolation or substantialisolation, initial growth of cells into colonies (incubation), editing(optionally induced, particularly in bacterial systems), then using cellgrowth—measured by colony size, concentration of metabolites or wasteproducts, or other characteristics that correlate with the rate ofgrowth of the cells—to select for cells that have been edited based onediting-induced growth delay. Selection may entail picking or selectingslow-growing cell colonies; alternatively, selection may entaileliminating (by, e.g., eradicating or removing) the faster-growing cellcolonies.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

As used herein, the terms “isolation” or “isolate” mean to separateindividual cells so that each cell (and the colonies formed from eachcell) will be separate from other cells; for example, a single cell in asingle microwell, or 100 single cells each in its own microwell.“Isolation” or “isolated cells” result in one embodiment, from a Poissondistribution in arraying cells. The terms “substantially isolated”,“largely isolated”, and “substantial isolation” mean cells are largelyseparated from one another, in small groups or batches. That is, when 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or up to 50—but preferably 10 or lesscells—are delivered to a microwell. “Substantially isolated” or “largelyisolated” result, in one embodiment, from a “substantial Poissondistribution” in arraying cells. With more complex libraries of edits—orwith libraries that may comprise lethal edits or edits withgreatly-varying fitness effects—it is preferred that cells be isolatedvia a Poisson distribution.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible. In the methods describedherein optionally the promoters driving transcription of the gRNAs isinducible.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418may be employed. In other embodiments, selectable markers include, butare not limited to human nerve growth factor receptor (detected with aMAb, such as described in U.S. Pat. No. 6,365,373); truncated humangrowth factor receptor (detected with MAb); mutant human dihydrofolatereductase (DHFR; fluorescent MTX substrate available); secreted alkalinephosphatase (SEAP; fluorescent substrate available); human thymidylatesynthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1;conjugates glutathione to the stem cell selective alkylator busulfan;chemoprotective selectable marker in CD34+cells); CD24 cell surfaceantigen in hematopoietic stem cells; rhamnose; human CAD gene to conferresistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drugresistance-1 (MDR-1; P-glycoprotein surface protein selectable byincreased drug resistance or enriched by FACS); human CD25 (IL-2α;detectable by MAb-FITC); Methylguanine-DNA methyltransferase (MGMT;selectable by carmustine); and Cytidine deaminase (CD; selectable byAra-C). “Selective medium” as used herein refers to cell growth mediumto which has been added a chemical compound or biological moiety thatselects for or against selectable markers.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, or in a nucleicacid (e.g., genome) of a cell or population of cells, in which a changeof at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The target sequence can be a genomic locus orextrachromosomal locus.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, YACs, BACs, mammalian synthetic chromosomes,and the like. As used herein, the phrase “engine vector” comprises acoding sequence for a nuclease—optionally under the control of aninducible promoter—to be used in the nucleic acid-guided nucleasesystems and methods of the present disclosure. The engine vector mayalso comprise, in a bacterial system, the λ Red recombineering system oran equivalent thereto, as well as a selectable marker. As used hereinthe phrase “editing vector” comprises a donor nucleic acid, including analteration to the target sequence which prevents nuclease binding at aPAM or spacer in the target sequence after editing has taken place, anda coding sequence for a gRNA optionally under the control of aninducible promoter (and preferably under the control of an induciblepromoter in bacterial systems). The editing vector may also comprise aselectable marker and/or a barcode. In some embodiments, the enginevector and editing vector may be combined; that is, the contents of theengine vector may be found on the editing vector.

Editing in Nucleic Acid-Guided Nuclease Genome Systems Generally

The present disclosure provides instruments, modules and methods fornucleic acid-guided nuclease genome editing that provide 1) enhancedobserved editing efficiency of nucleic acid-guided nuclease editingmethods, and 2) improvement in screening for and detecting cells whosegenomes have been properly edited, including high-throughput screeningtechniques. In current protocols employing nuclease systems, bulkculture of cells with constitutively-expressed nuclease componentstypically are used to drive high-efficiency editing. However, pooled ormultiplex formats can lead to selective enrichment of cells that are notedited due to the lack of double-strand DNA breaks that occur duringediting.

Presented herein are methods that take advantage of isolation(separating cells and growing them into clonal colonies) and eithernormalization of cell colonies or cherry picking of slow-growingcolonies. Isolation or substantial isolation, incubation, followed byediting (optionally with a gRNA under the control of an induciblepromoter) and normalization overcomes growth bias from unedited cells,and substituting cherry picking for normalization allows for directselection of edited cells. The instruments, modules, and methods may beapplied to all cell types including, archaeal, prokaryotic, andeukaryotic (e.g., yeast, fungal, plant and animal) cells.

The instruments, modules, and methods described herein employ editingcassettes comprising a guide RNA (gRNA) sequence covalently linked to adonor DNA sequence where, particularly in bacterial systems, the gRNAoptionally is under the control of an inducible promoter (e.g., theediting cassettes are CREATE cassettes; see U.S. patent Ser. No.9/982,278, issued 29 May 2019 and Ser. No. 10/240,167, issued 26 Mar.2019; Ser. No. 10/266,849, issued 23 Apr. 2019; and U.S. Ser. No.15/948,785, filed 9 Apr. 2018; Ser. No. 16/275,439, filed 14 Feb. 2019;and Ser. No. 16/275,465, filed 14 Feb. 2019, all of which areincorporated by reference in their entirety). The disclosed methodsallow for cells to be transformed, substantially isolated, grown forseveral doublings (e.g., incubation), after which editing is allowed.The isolation process effectively negates the effect of unedited cellstaking over the cell population. The combination of substantiallyisolating cells, then allowing for initial growth followed by optionallyinducing transcription of the gRNA (and optionally the nuclease) andeither normalization of cell colonies or cherry picking cells leads to2-250×, 10-225×, 25-200×, 40-175×, 50-150×, 60-100×, or 50-100× gains inidentifying edited cells over prior art methods and allows forgeneration of arrayed or pooled edited cells comprising cell librarieswith edited genomes. Additionally, the methods may be leveraged tocreate iterative editing systems to generate combinatorial libraries ofcells with two to many edits in each cellular genome. Optionally usinginducible gRNA constructs (and in some embodiments, inducible nucleaseconstructs) provides “pulsed” exposure of the cells to active editingcomponents, which 1) allows for the cells to be arrayed (e.g., largelyisolated) prior to initiation of the editing procedure, 2) decreasesoff-target activity, 3) allows for identification of rare cell edits,and 4) enriches for edited cells or permit high-throughput screeningapplications to identify editing activity using cell growth as a proxyfor editing, by, e.g., measuring optical density, colony size, ormetabolic by-products or other characteristics thereby enriching theedited cell population.

The instruments, compositions and methods described herein improveediting systems in which nucleic acid-guided nucleases (e.g., RNA-guidednucleases) are used to edit specific target regions in an organism'sgenome. A nucleic acid-guided nuclease complexed with an appropriatesynthetic guide nucleic acid in a cell can cut the genome of the cell ata desired location. The guide nucleic acid helps the nucleic acid-guidednuclease recognize and cut the DNA at a specific target sequence. Bymanipulating the nucleotide sequence of the guide nucleic acid, thenucleic acid-guided nuclease may be programmed to target any DNAsequence for cleavage as long as an appropriate protospacer adjacentmotif (PAM) is nearby. In certain aspects, the nucleic acid-guidednuclease editing system may use two separate guide nucleic acidmolecules that combine to function as a guide nucleic acid, e.g., aCRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In otheraspects, the guide nucleic acid may be a single guide nucleic acid thatincludes both the crRNA and tracrRNA sequences or a single guide nucleicacid that does not require a tracrRNA.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA is encoded bya DNA sequence on a polynucleotide molecule such as a plasmid, linearconstruct, or resides within an editing cassette and isoptionally—particularly in bacterial systems—under the control of aninducible promoter.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence (the portion of theguide nucleic acid that hybridizes with the target sequence) is about ormore than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides inlength. In some embodiments, a guide sequence is less than about 75, 50,45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guidesequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or20 nucleotides in length.

In the present methods and compositions, the guide nucleic acid isprovided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript. Alternatively, the guide nucleic acids may be transcribedfrom two separate sequences. The guide nucleic acid can be engineered totarget a desired target DNA sequence by altering the guide sequence sothat the guide sequence is complementary to the target DNA sequence,thereby allowing hybridization between the guide sequence and the targetDNA sequence. In general, to generate an edit in the target DNAsequence, the gRNA/nuclease complex binds to a target sequence asdetermined by the guide RNA, and the nuclease recognizes a protospaceradjacent motif (PAM) sequence adjacent to the target sequence. Thetarget sequence can be any polynucleotide (either DNA or RNA) endogenousor exogenous to a prokaryotic or eukaryotic cell, or in vitro. Forexample, the target sequence can be a polynucleotide residing in thenucleus of a eukaryotic cell. A target sequence can be a sequenceencoding a gene product (e.g., a protein) and/or a non-coding sequence(e.g., a regulatory polynucleotide, an intron, a PAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid; that is, the editing cassette may be a CREATEcassette (see, e.g., U.S. patent Ser. No. 9/982,278, issued 29 May 2019and Ser. No. 10/240,167, issued 26 Mar. 2019; Ser. No. 10/266,849,issued 23 Apr. 2019; and U.S. Ser. No. 15/948,785, filed 9 Apr. 2018;Ser. No. 16/275,439, filed 14 Feb. 2019; and Ser. No. 16/275,465, filed14 Feb. 2019, all of which are incorporated by reference in theirentirety). The guide nucleic acid and the donor nucleic acid may be andtypically are under the control of a single (optionally inducible)promoter. Alternatively, the guide nucleic acid may not be part of theediting cassette and instead may be encoded on the engine or editingvector backbone. For example, a sequence coding for a guide nucleic acidcan be assembled or inserted into a vector backbone first, followed byinsertion of the donor nucleic acid. In other cases, the donor nucleicacid can be inserted or assembled into a vector backbone first, followedby insertion of the sequence coding for the guide nucleic acid. In yetother cases, the sequence encoding the guide nucleic acid and the donornucleic acid (inserted, for example, in an editing cassette) aresimultaneously but separately inserted or assembled into a vector. Inyet other embodiments and preferably, the sequence encoding the guidenucleic acid and the sequence encoding the donor nucleic acid are bothincluded in the editing cassette.

The target sequence is associated with a PAM, which is a shortnucleotide sequence recognized by the gRNA/nuclease complex. The precisePAM sequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence. Engineering of thePAM-interacting domain of a nucleic acid-guided nuclease may allow foralteration of PAM specificity, improve target site recognition fidelity,decrease target site recognition fidelity, and increase the versatilityof a nucleic acid-guided nuclease. In certain embodiments, the genomeediting of a target sequence both introduces a desired DNA change to atarget sequence, e.g., the genomic DNA of a cell, and removes, mutates,or renders inactive a proto-spacer (PAM) region in the target sequence;that is, the donor DNA often includes an alteration to the targetsequence that prevents binding of the nuclease at the PAM in the targetsequence after editing has taken place. Rendering the PAM at the targetsequence inactive precludes additional editing of the cell genome atthat target sequence, e.g., upon subsequent exposure to a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acid inlater rounds of editing. Thus, cells having the desired target sequenceedit and an altered PAM can be selected using a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid complementary tothe target sequence. Cells that did not undergo the first editing eventwill be cut rendering a double-stranded DNA break, and thus will notcontinue to be viable. The cells containing the desired target sequenceedit and PAM alteration will not be cut, as these edited cells no longercontain the necessary PAM site and will continue to grow and propagate.

The range of target sequences that nucleic acid-guided nucleases canrecognize is constrained by the need for a specific PAM to be locatednear the desired target sequence. As a result, it often can be difficultto target edits with the precision that is necessary for genome editing.It has been found that nucleases can recognize some PAMs very well(e.g., canonical PAMs), and other PAMs less well or poorly (e.g.,non-canonical PAMs). Because the methods disclosed herein allow foridentification of edited cells in a large background of unedited cells,the methods allow for identification of edited cells where the PAM isless than optimal; that is, the methods for identifying edited cellsherein allow for identification of edited cells even if editingefficiency is very low. Additionally, the present methods expand thescope of target sequences that may be edited since edits are morereadily identified, including cells where the genome edits areassociated with less functional PAMs.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryoticcells can be yeast, fungi, algae, plant, animal, or human cells.Eukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, mouse, rat,rabbit, dog, or non-human mammal including non-human primate. The choiceof nucleic acid-guided nuclease to be employed depends on many factors,such as what type of edit is to be made in the target sequence andwhether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.As with the guide nucleic acid, the nuclease may be encoded by a DNAsequence on a vector (e.g., the engine vector) and be under the controlof a constitutive or an inducible promoter. Again, at least one of andpreferably both of the nuclease and guide nucleic acid are under thecontrol of an inducible promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., vector or editing (CREATE) cassette) asthe guide nucleic acid. The donor nucleic acid is designed to serve as atemplate for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 30, 35, 40, 45, 50,75, 100, 150, 200, 500, 1000, 2500, 5000 nucleotides or more in length.In certain preferred aspects, the donor nucleic acid can be provided asan oligonucleotide of between 40-300 nucleotides, more preferablybetween 50-250 nucleotides. The donor nucleic acid comprises a regionthat is complementary to a portion of the target sequence (e.g., ahomology arm). When optimally aligned, the donor nucleic acid overlapswith (is complementary to) the target sequence by, e.g., about 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In manyembodiments, the donor nucleic acid comprises two homology arms (regionscomplementary to the target sequence) flanking the mutation ordifference between the donor nucleic acid and the target template. Thedonor nucleic acid comprises at least one mutation or alterationcompared to the target sequence, such as an insertion, deletion,modification, or any combination thereof compared to the targetsequence.

Often the donor nucleic acid is provided as an editing cassette, whichis inserted into a vector backbone where the vector backbone maycomprise a promoter driving transcription of the gRNA and the donornucleic acid. Moreover, there may be more than one, e.g., two, three,four, or more guide nucleic acid/donor nucleic acid cassettes insertedinto an engine vector, where the guide nucleic acids are under thecontrol of separate, different promoters, separate, like promoters, orwhere all guide nucleic acid/donor nucleic acid pairs are under thecontrol of a single promoter. (See, e.g., U.S. Ser. No. 16/275,465,filed 14 Feb. 2019, drawn to multiple CREATE cassettes.) The promoterdriving transcription of the gRNA and the donor nucleic acid (or drivingmore than one gRNA/donor nucleic acid pair) is optionally an induciblepromoter (and in bacterial systems is preferably an inducible promoter)and the promoter driving transcription of the nuclease is optionally aninducible promoter as well.

Inducible editing is advantageous in that substantially or largelyisolated cells can be grown for several to many cell doublings beforeediting is initiated, which increases the likelihood that cells withedits will survive, as the double-strand cuts caused by active editingare largely toxic to the cells. This toxicity results both in cell deathin the edited colonies, as well as a lag in growth for the edited cellsthat do survive but must repair and recover following editing. However,once the edited cells have a chance to recover, the size of the coloniesof the edited cells will eventually catch up to the size of the coloniesof unedited cells (e.g., the process of “normalization” or growingcolonies to “terminal size”; see, e.g., FIG. 1B described infra).

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a target sequence—oneor more PAM sequence alterations that mutate, delete or render inactivethe PAM site in the target sequence. The PAM sequence alteration in thetarget sequence renders the PAM site “immune” to the nucleic acid-guidednuclease and protects the target sequence from further editing insubsequent rounds of editing if the same nuclease is used.

The editing cassette also may comprise a barcode. A barcode is a uniqueDNA sequence that corresponds to the donor DNA sequence such that thebarcode can identify the edit made to the corresponding target sequence.The barcode can comprise greater than four nucleotides. In someembodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or genome-wide libraries ofdonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid designis associated with a different barcode, or, alternatively, eachdifferent cassette molecule is associate with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes a nucleic acid-guided nuclease comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineerednuclease comprises NLSs at or near the amino-terminus, NLSs at or nearthe carboxy-terminus, or a combination.

Exemplary Workflows for Editing, Enrichment, and Selection of EditedCells

The methods described herein provide enhanced observed editingefficiency of nucleic acid-guided nuclease editing methods as the resultof a combination of isolation or substantial isolation, initial cellgrowth (incubation), editing, and either normalization of the resultingcell colonies or cherry picking slow-growing cell colonies. Thecombination of the isolation or substantial isolation, initial cellgrowth, editing and normalization processes overcomes the growth bias infavor of unedited cells—and the fitness effects of editing (includingdifferential editing rates)—thus allowing all cells “equal billing” withone another. The combination of isolation or substantial isolation,initial cell growth, editing, and cherry picking allows for directselection of edited colonies of cells. The result of the methodsdescribed herein is that even in nucleic acid-guided nuclease systemswhere editing is not optimal—such as in systems where non-canonical PAMsare targeted—there is an increase in the observed editing efficiency;that is, edited cells can be identified even in a large background ofunedited cells. Observed editing efficiency can be improved up to 80% ormore. Isolating, incubating, editing, and normalization of cell coloniesor cherry picking of cell colonies leads to 2-250×, 10-225×, 25-200×,40-175×, 50-150×, 60-100×, or 5-100× gains in identifying edited cellsover prior art methods and allows for the generation of arrayed orpooled edited cells comprising genome libraries. Additionally, theinstruments, modules and methods may be leveraged to create iterativeediting systems to generate combinatorial libraries, identify rare celledits, and enable high-throughput enrichment applications to identifyediting activity.

FIG. 1A shows simplified flow charts for two alternative exemplarymethods 100 a and 100 b for isolating cells for enrichment (100 a) andfor cherry picking (100 b). Looking at FIG. 1A, method 100 begins bytransforming cells 110 with the components necessary to perform nucleicacid-guided nuclease editing. For example, the cells may be transformedsimultaneously with separate engine and editing vectors; the cells mayalready be transformed with an engine vector expressing the nuclease(e.g., the cells may have already been transformed with an engine vectoror the coding sequence for the nuclease may be stably integrated intothe cellular genome) such that only the editing vector needs to betransformed into the cells; or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing.

A variety of delivery systems can be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a host cell 110. These delivery systems include the use of yeastsystems, lipofection systems, microinjection systems, biolistic systems,virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acidconjugates, virions, artificial virions, viral vectors, electroporation,cell permeable peptides, nanoparticles, nanowires, exosomes.Alternatively, molecular trojan horse liposomes may be used to delivernucleic acid-guided nuclease components across the blood brain barrier.Of interest, particularly in the context of an automated multi-modulecell editing instrument is the use of electroporation, particularlyflow-through electroporation (either as a stand-alone instrument or as amodule in an automated multi-module system) as described in, e.g., U.S.Ser. No. 16/147,120, filed 28 Sep. 2018; Ser. No. 16/147,353, filed 28Sep. 2018; Ser. No. 16/147,865, filed 30 Sep. 2018; and Ser. No.16/147,871, filed 30 Sep. 2018. If the solid wall isolation orsubstantial isolation/incubation/editing and normalization module is onemodule in an automated multi-module cell editing instrument as describedherein infra, the cells are likely transformed in an automated celltransformation module.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are substantially orlargely isolated 120; that is, the cells are diluted (if necessary) in aliquid culture medium so that the cells, when delivered to a substratefor isolation, are substantially separated from one another and can formcolonies that are substantially separated from one another. For example,if a solid wall device is used (described infra in relation to FIGS.3A-3E and 4A-4Z), the cells are diluted such that when delivered to thesolid wall device the cells fill the microwells of the solid wall devicein a Poisson or substantial Poisson distribution. In one example(illustrated in FIG. 3A), isolation is accomplished when an average of ½cell is delivered to each microwell; that is, where some microwellscontain one cell and other microwells contain no cells. Alternatively, asubstantial Poisson distribution of cells occurs when two to several (2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or up to 50, but preferably 10 or less,or preferably 5 or less) cells are delivered to a microwell (illustratedin FIG. 3B).

Once the cells have been isolated or substantially or largely isolated120, the cells are allowed to grow to, e.g., between 2 and 130, orbetween 5 and 120, or between 10 and 100 doublings, establishing clonalcolonies 130. After colonies are established, editing proceeds 140. Insome systems, for example, bacterial systems, an inducible system isused where at least the gRNA is under the control of an induciblepromoter (see FIG. 1C described in detail infra). Once editing iscomplete, in enrichment method 100 a the cells are grown into coloniesof terminal size 150; that is, the colonies arising from thesubstantially or largely isolated cells are grown into colonies to apoint where cell growth has peaked and is normalized or saturated forboth edited and unedited cells. Normalization occurs as the nutrients inthe medium around a growing cell colony are depleted and/or cell growthfills the microwells and further growth is physically constrained. Theterminal-size colonies are pooled 160 by, e.g., scraping the coloniesoff a plate comprising solid medium or other substrate or by flushingclonal cell colonies from microwells in a solid wall device or module topool the cells from the normalized cell colonies. Again, becauseisolation or substantial isolation overcomes growth bias from uneditedcells or cells exhibiting fitness effects as the result of edits made,isolation or substantial isolation, incubation, editing, andnormalization alone enriches the total population of cells with cellsthat have been edited; that is, isolation or substantial isolationcombined with incubation, editing and normalization (e.g., growingcolonies to terminal size) allows for high-throughput enrichment ofedited cells.

Method 100 b shown in FIG. 1A is similar to the method 100 a in thatcells of interest are transformed 110 with the components necessary toperform nucleic acid-guided nuclease editing. As described above, thecells may be transformed simultaneously with both the engine and editingvectors, the cells may already be expressing the nuclease (e.g., thecells may have already been transformed with an engine vector or thecoding sequence for the nuclease may be stably integrated into thecellular genome) such that only the editing vector needs to betransformed into the cells, or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing. Further, if the isolation orsubstantial isolation of cells, incubation, editing, and eithernormalization or cherry-picking module (“solid wallisolation/incubation/normalization module” or “SWIIN”) is one module inan automated multi-module cell editing instrument (such as that shown inFIGS. 5A-5D and described below), cell transformation may be performedin an automated flow-through transformation module as described inrelation to FIGS. 8A-8E below.

After the cells are transformed 110 with the components necessary toperform nucleic acid-guided nuclease editing, the cells are diluted (ifnecessary) in liquid medium so that the cells, when delivered to anisolation device or module, are separated from one another and can formcolonies that are separated from one another. For example, if a solidwall device is used (described in relation to FIGS. 3A-3J and 4A-4Y) thecells are diluted such that when delivered to the solid wall device, thecells fill the microwells of the solid wall device in a Poisson orsubstantial Poisson distribution.

Once the cells have been substantially or largely isolated 120, thecells grown to, e.g., between 2 and 150, or between 5 and 120, orbetween 10 and 100 doublings, establishing clonal colonies 130. Aftercolonies are established, editing is allowed 140. In some embodimentssuch as in bacteria, editing is induced by, e.g., activating induciblepromoters that control transcription of the gRNA and optionally thenuclease and a recombineering system. Once editing begins 140, many ofthe edited cells in the clonal colonies die due to the double-strand DNAbreaks that occur during the editing process; however, in a percentageof edited cells, the genome is edited and the double-strand break isproperly repaired. When allowed to recover, these edited cells startgrowing and re-establish cell populations within each isolated partitionor colony; however, the growth of edited colonies tends to lag behindthe growth of clonal colonies where an edit has not taken place (e.g.,cell “escapees”). If growth of these colonies is monitored 170, thesmall or slow-growing colonies (edited cells) may be identified and thenselected or cherry picked 180 based on the observable size differentialsof the colonies.

FIG. 1B is a plot of OD versus time for unedited cells (solid line)versus edited cells (dashed line). FIG. 1B shows how normalization ofedited and non-edited colonies takes place. Note that the OD (e.g.,growth) of the edited cells lags behind the unedited cells initially,but eventually catches up due, e.g., to unedited cells exhausting thenutrients in the medium, becoming physically constrained within amicrowell or other confined growth area (such as a droplet), orotherwise exiting log-phase growth. The cell colonies are allowed togrow long enough for the growth of the edited colonies to catch up with(approximate the size of, e.g., number of cells in) the uneditedcolonies.

FIG. 1C depicts an exemplary inducible expression system such as used inbacteria—in this example, the pL inducible system—for regulating gRNAactivity. At the top of FIG. 1C there is shown a portion of an exemplaryengine vector 111 comprising an origin of replication 112, a promoter114 driving expression of the c1857 repressor gene 116, and a first pLpromoter 118 driving expression of a nuclease 121. At the top of FIG. 1Cthere is also seen a portion of an exemplary editing vector 131,comprising an origin of replication 132, and a second pL promoter 134driving transcription of an editing cassette 136 (e.g., a CREATEcassette) which includes a coding sequences for both a gRNA and a donorDNA. The middle illustration of FIG. 1C depicts the product 124 of thec1857 repressor gene 116 on the engine vector 111 actively repressingthe first pL promoter 118 driving transcription of the nuclease 121 andthe second pL promoter 134 driving transcription of the editing cassette136 on the editing vector 131. Finally, the bottom illustration of FIG.1C depicts the protein product 126 of the c1857 repressor gene 116 onthe engine vector 111 unfolding/degrading due to increased temperature.The unfolded or degraded protein product 126 cannot bind first pLpromoter 118 or second pL promoter 134; thus, pL promoter 118 is activeand drives transcription of the nuclease 121 on engine vector 111 and pLpromoter 134 is active and drives transcription of the editing cassette136 on the editing vector 131.

In FIG. 1C, transcription of both the nuclease and the gRNA are undercontrol of a pL promoter; however, in other embodiments, differentinducible promoters may be used to drive transcription of the nucleaseand gRNA or in some embodiments only the gRNA is under control of aninducible promoter. For example, the pL and pBAD promoters are shown inrelation to the exemplary bacterial engine and editing vectors in FIGS.11A and 11B, and a number of gene regulation control systems have beendeveloped for the controlled expression of genes in plant, microbe andanimal cells, including mammalian cells. These systems include thetetracycline-controlled transcriptional activation system(Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard and Gossen,PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system(Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur etal., Strategies 5(3):70-72 (1992); and U.S. Pat. No. 4,833,080), theecdysone-inducible gene expression system (No et al., PNAS,93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al.,BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible geneexpression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) aswell as others. However, the pL promoter is a particularly usefulinducible promoter because the pL promoter is activated by an increasein temperature, to, e.g., 42° C., and deactivated by returning thetemperature to, e.g., 30° C. With other inducible systems that areactivated by the presence or absence of a particular molecular compound,activating the inducible promoter requires addition of or removal of amolecular compound from the culture medium, thus requiring liquidhandling, medium exchange, wash steps and the like.

FIG. 2A depicts a standard, conventional, prior art protocol 200 forperforming nucleic acid-guided nuclease genome editing whereconstitutively-expressed nuclease components typically are used to drivehigh efficiency editing. In FIG. 2A, a library or collection of editingvectors 202 is introduced 203 (e.g., electroporated) into cultured cells204 that comprise a coding sequence for a nuclease under the control ofa constitutive or inducible promoter. In some embodiments, the codingsequence for the nuclease is contained on an “engine vector”, althoughin other embodiments the coding sequence for the nuclease may beintegrated into the cell genome. In yet another alternative, thecomponents of the engine and editing vectors may be combined. Theediting vectors 202 comprise an editing sequence comprising a sequencefor a desired edit in a nucleic acid sequence endogenous to the cell aswell as an optional PAM-altering sequence (most often a sequence thatdisables the PAM at the target sequence in the genome), a codingsequence for a gRNA under the control of a constitutive promoter, and aselectable marker. Once the cells have been transformed with the editingvectors, the cells are plated 205 on selective medium 206 to select forcells that have both the engine and the editing vectors and the cellsare grown until colonies form. Cells are then picked 207 from thecolonies and grown in, e.g., 96-well plates 208 to be prepared forgenome or plasmid sequencing. The cells that grow on plate 206 with theselective medium should comprise both the engine and editing vectors;however, it is likely that in some cells the nucleic acid-guided editingcomponents may be non-functional. In pooled or multiplex formats, thereis likely to be selective enrichment of the cells with a non-functionalediting system since un-edited cells are not subjected to thedouble-strand DNA breaks that occur during editing. The strong selectionfor cells with non-functional nucleic acid-guided nuclease editingsystems leads to a disproportionate representation of non-edited cellsin the final cell population and compromises the integrity of librariesgenerated via multiplexed nuclease editing systems.

FIGS. 2B-2F depict improved protocols for performing nucleic acid-guidednuclease genome editing. FIG. 2B depicts a first embodiment 201 of animproved protocol for performing nucleic acid-guided nuclease genomeediting using an optional inducible promoter to drive expression of thegRNA. In FIG. 2B, a library or collection of editing vectors 202 isintroduced 203 (e.g., electroporated) into cultured cells 204 thatcomprise a coding sequence for a nuclease under the control of aconstitutive or inducible promoter; however, in bacteria the tightestregulation of the nucleic acid-guided nuclease system is achieved byusing an inducible promoter to drive expression of the nuclease, thus itis preferred that an inducible promoter is used to drive transcriptionof the nuclease. In some embodiments, the coding sequence for thenuclease is contained on an “engine plasmid” (most often along with,e.g., a selectable marker) that has already been transformed into thecells, although in other embodiments the coding sequence for thenuclease may be integrated into the genome of the cells. In yet otherembodiments such as in yeast, the coding sequence for the nuclease maybe located on the editing vector (that is, a combined engine and editingvector). The editing vectors 202 comprise an editing sequence, whichoptionally includes a PAM-altering sequence (most often a sequence thatdisables the PAM at the target site in the genome), a coding sequencefor a gRNA under the control of an inducible promoter, and a selectablemarker.

Once the cells 204 have been transformed with the editing vectors, thecells are plated 205 on selective medium to select for cells that haveboth the engine and the editing vectors and grown until colonies 206form. Cells are then picked 207 from the colonies and grown overnightin, e.g., first 96-well plate 208 in medium that selects for both theengine and editing vectors. In a next step 209, the cells from the first96-well plate 208 are replicated into a second 96-well plate 210 intomedium containing an additional selective component such as, e.g.,arabinose, to drive strong induction of the λ red recombineering system(the homologous repair machinery). The inducible pBAD promoter—inducedby the presence of arabinose in the growth medium—controls the λ redrecombineering system and is described in relation to FIG. 11A below.Again, though a recombineering system is exemplified here in a bacterialediting system, recombineering systems generally are not needed ineukaryotic editing systems. After initial cell growth at 30° C., cuttingand editing of the cellular genome is induced by increasing thetemperature to 42° C. in the second 96-well plate 210 for, e.g.,one-half to approximately two hours (depending on cell type), toactivate the pL inducible promoters which drive transcription of thenuclease and the gRNA. Following induction of cutting and editing—e.g.,for approximately 2 hours—the temperature is returned to 30° C. to allowthe cells to recover.

In addition, in a step 211 cells from the first 96-well plate 208 arereplicated into a third 96-well plate 214 into medium that does notcontain the selective component that induces the homologous repairmachinery (here, arabinose, such that the λ Red recombineering system isnot activated). However, the third 96-well plate 214—aside from nothaving arabinose added to the growth medium—is subjected to the sameconditions as the second 96-well plate; that is, initial cell growth at30° C., increasing the temperature to 42° C. for, e.g., two hours toactivate the pL inducible promoter driving the expression of thenuclease and the gRNA, then reducing the temperature to 30° C. to allowthe cells to recover. As an alternative to 96-well plates 210 and 214,shown are two agar plates 212 and 216, where cells from plate 206 arearrayed. Plate 212 corresponds to the second 96-well plate 210,containing growth medium with arabinose, and plate 216 corresponds tothe third 96-well plate 214, containing growth medium without arabinose.Note that as an alternative to performing the method depicted in FIG. 2Bwith +/− arabinose, one may perform the method with −/+ temperatureinduction of cutting. For example, in a +/− temperature inductionexperiment, plate 210 would be the uninduced culture plate (no temp) andculture plate 214 would be the temperature-induced culture plate. Thesame overall interpretation applies; however, cut activity is isolatedto avoid having to deconvolute cut from paste.

Looking at the second and third 96-well plates (210 and 214,respectively) and at plates 212 and 216, colonies of cells can be seen.Looking first at second 96-well plate 210, there are two types ofcolonies: 226 (white wells) and 228 (hatched wells). Looking at third96-well plate 214, there are two types of colonies: 220 (black wells)and 226 (white wells). Replicate plating of cells from the first 96-wellplate into the second and third 96-well plates with differential mediaallows for functional deconvolution of the resulting cell populations.In third 96-well plate 214, cells were cultured in a medium withoutarabinose. With no arabinose, the λ Red recombineering system is notactive, and cells that have active gRNA and nuclease expression (inducedby increasing the temperature of the cultured cells to 42° C.) are notviable due to the double-strand cuts made by the active gRNA andnuclease yet without repair by the λ Red recombineering system. Thesecell colonies 222 are denoted by white wells. The cell colonies 220denoted by black wells represent cells that have inactive gRNAs, suchthat the genomes of cells in these colonies are not cut by the gRNA andnuclease complex. Inactive gRNAs occur at a frequency of approximately2-15% in a typical cell editing experiment, and typically are the resultof errors in the guide sequence portion (homology portion) of the gRNA.

In second 96-well plate 210, the cells were cultured in a medium witharabinose, thereby activating the λ Red recombineering system. Cellsthat have active gRNA and nuclease expression (induced by increasing thetemperature of the cultured cells to 42° C.) are cut, edited, and the λRed recombineering system repairs the double-strand DNA breaks and thusthese cells are viable. However, also viable are cells with inactivegRNAs (or less frequently, inactive nucleases). All viable colonies inplate 210 are denoted by hatched wells 228. Finally, cells that haveactive gRNAs but are not edited appropriately (due to, e.g., an inactiverecombineering system) are denoted by white wells 226. By comparing thesecond 96-well plate to the third 96-well plate, the function of thenucleic acid-guided nuclease system in each colony can be determined.For example, if colonies of cells grow without arabinose (plate 214,black wells 220) and with arabinose (plate 210, hatched wells 228), thecells must not have an active gRNA. If the cells had an active gRNA,they would not have been viable in medium without arabinose (e.g.,without an active λ Red recombineering system). If colonies of cellsfail to grow in medium without arabinose (plate 214, white wells 226)and do grow when arabinose is added to the culture medium (plate 210,hatched wells 228), the cells in these wells comprise an active nucleicacid-guided nuclease system—with active gRNA and nuclease components—andare very likely to have been properly edited. Finally, if the coloniesof cells fail to grow either without arabinose (plate 214, white wells226) or with arabinose (plate 210, white wells 226), the gRNA is active,but the cells are not able to repair the cut for some reason. Thus, themethod depicted in FIG. 2B allows for identification of edited cellcolonies via functional deconvolution of the various components of theediting “machinery” in one experiment.

In plates 212 and 216 the phenotypic readout is the same as in 96-wellplates 210 and 214. Like 96-well plate 210, the medium in culture dish212 contains arabinose, and like 96-well plate 214, the medium inculture dish 216 does not contain arabinose. Thus, in culture dish 216cells with inactive gRNAs grow, as there is no edit (double-strandbreak) to repair. However, in cells with an active gRNA, editing takesplace but there is no active repair machinery to repair the edits, andthe cells are not viable and do not form colonies. In culture dish 212that contains arabinose, cells that have inactive gRNAs form colonies,as do cells that have active gRNAs but are properly edited. Cells thatare not viable for whatever reason do not form colonies. As with 96-wellplates 210 and 214, comparison of culture dishes 212 and 214 allow oneto deconvolute the various components of the editing machinery. If cellsgrow on both culture dishes 212 and 214, the gRNA is likely inactive. Ifcells grow on culture dish 212 but not on culture dish 214, the gRNA islikely active and proper editing has taken place. If cells fail to growon either culture dish 212 or 214, the cells likely have an active gRNAbut the edit is not repaired properly.

Thus, the method 201 depicted in FIG. 2B allows for identification ofcells with nonfunctional gRNAs that, due to the lack of double-strandDNA breaks, have a growth advantage. Most importantly, the method 201depicted in FIG. 2B allows for identification of cells that have beenproperly edited. An aliquot of the colonies 228 from either second96-well plate 210 or agar plate 212—the colonies confirmed to have anactive gRNA—can be picked and sequenced to confirm editing. 96-wellplate 210 or agar plate 212 may be retained so that once proper editingis confirmed by, e.g., sequencing, one can go back to plate 210 or 212and retrieve the properly-edited cells. Plates 210 and 212 may bereferred to, e.g., as “cell hotels” or “cell repositories.” Note thatthe method 201 depicted in FIG. 2B first substantially or largelyisolates the cells by plating them on selective medium at an appropriatedilution such that single cells form single colonies. Again, isolationor substantial isolation overcomes growth the bias that ischaracteristic of unedited cells. The method then allows for functionaldeconvolution of the different colonies by replica plating colonies in96-well microtiter plates under different section media to allowpositive identification of edited colonies. A specific protocol for thisexemplary method is described in Example 6 below.

FIG. 2C depicts a second exemplary embodiment of an improved protocol240 for performing nucleic acid-guided nuclease genome editing using aninducible promoter in a bacterial system to drive expression of thegRNA, and, preferably, the nuclease as well. In FIG. 2C as in FIG. 2B, alibrary or collection of editing vectors 202 is introduced 203 (e.g.,electroporated) into cultured cells 204 that comprise a coding sequencefor a nuclease under the control of a constitutive or induciblepromoter; however, the tightest regulation of the nucleic acid-guidednuclease system is achieved by using an inducible promoter to driveexpression of the nuclease and thus is preferred. Also like FIG. 2B, insome embodiments, the coding sequence for the nuclease is contained onan “engine plasmid” (most often along with, e.g., a selectable marker)that has already been transformed into the cells, although in otherembodiments, the coding sequence for the nuclease may be integrated intothe genome of the cells. In yet other embodiments such as in yeast, thecoding sequence for the nuclease may be located on the editing vector(that is, a combined engine and editing vector). The editing vectors 202comprise an editing sequence with a desired edit vis-à-vis an endogenousnucleic acid sequence in the cell along with a PAM-altering sequence(most often a sequence that disables the PAM at the target site in thegenome), a coding sequence for a gRNA under the control of, preferably,an inducible promoter, and a selectable marker.

Once the cells 204 have been transformed with the editing vectors, thecells are plated 235 on selective medium on substrate or plate 236 toselect for cells that have both the engine and the editing vectors. Thecells are diluted before plating such that the cells are substantiallyor largely isolated—separated enough so that they and the colonies theyform are separated from other cell colonies—and the cells are then grown237 on plate or substrate 236 until colonies 238 begin to form. Thecells are allowed to grow at, e.g., 30° C. for, e.g., between 2 and 150,or between 5 and 120, or between 10 and 50 doublings, establishingclonal colonies. This initial growth of cells is to accumulate enoughclonal cells in a colony to survive induction of editing. Once coloniesare established, cutting and editing of the cellular genome is inducedby inducing or activating the promoters driving at least the gRNA (andin some instances the nuclease as well), and the λ Red recombineeringsystem (if present). If the λ Red recombineering system is present andunder the control of an inducible promoter, preferably this induciblepromoter is different from the inducible promoter driving transcriptionof the gRNA and nuclease and is activated (induced) before induction ofthe gRNA and nuclease. The λ Red recombineering system works as the“band aid” or repair system for double-strand breaks in bacteria, and insome species of bacteria must be present for the double-strand breaksthat occur during editing to resolve. The λ Red recombineering systemmay be under the control of, e.g., a pBAD promoter. The pBAD promoter,like the pL promoter, is an inducible promoter; however, the pBADpromoter is regulated (induced) by the addition of arabinose to thegrowth medium. Thus, if there is arabinose contained in the selectivemedium of substrate or plate 236, the λ Red recombineering system willbe activated when the cells are grown 237. As for induction of editing239, if transcription of the gRNA and nuclease are both under control ofthe pL promoter, transcription of the gRNA and nuclease is induced byincreasing the temperature to 42° C. for, e.g., a half-hour to two hours(or more, depending on the cell type), which activates the pL induciblepromoter. Following induction of cutting and editing and a two-hour 42°C. incubation, the temperature is returned to 30° C. to allow the cellsto recover and to disable the pL promoter system.

Once the cells have recovered and are growing at 30° C., the cells aregrown on substrate or plate 236 into colonies of terminal size 244; thatis, the colonies arising from the substantially or largely isolatedcells are grown into colonies to a point where cell growth has peakedand becomes normalized (e.g., saturated) for both edited and uneditedcells. As described supra, normalization occurs as the nutrients in themedium around a growing cell colony are depleted and/or cell growthfills the microwells or is otherwise constrained; that is, the cells arein senescence. The terminal-size colonies are then pooled 241 by, e.g.,scraping the colonies off the substrate or plates (or by, e.g., flushingthe clonal cell colonies from microwells in a solid wall device) to pool280 the cells from the normalized cell colonies. Note that the method240 illustrated and described for FIG. 2C utilizes isolation orsubstantial isolation, initial growth (e.g., incubation), editing,normalization and pooling of the resulting cell colonies. Again, becauseisolation or substantial isolation overcomes growth bias from uneditedcells or cells exhibiting fitness effects as the result of edits made,the combination of isolation or substantial isolation, incubation,editing, and normalization enriches the total population of cells withcells that have been edited. Note, however, unlike the methods 201, 250depicted in FIG. 2B and FIG. 2D respectively, method 240 in FIG. 2Ctakes no steps to deconvolute the editing “machinery.”

FIG. 2D depicts a third exemplary embodiment of an improved protocol 250for performing nucleic acid-guided nuclease genome editing using aninducible promoter to drive expression of the gRNA, and in someembodiments and preferably, the nuclease as well. FIG. 2D depicts aprotocol 250 for activity-based error correction and re-arraying. InFIG. 2D as in FIGS. 2B and 2C, a library or collection of editingvectors 202 is introduced 203 (e.g., electroporated) into cultured cells204 that comprise a coding sequence for a nuclease under the control ofa constitutive or inducible promoter (preferably inducible), eithercontained on an “engine plasmid” (e.g., along with, e.g., a selectablemarker) that has already been transformed into the cells, or integratedinto the genome of the cells. Alternatively, such as in yeast the codingsequence for the nuclease may be located on the editing vector. Theediting vectors 202 comprise an editing sequence, a PAM-alteringsequence (most often a sequence that disables the PAM at the target sitein the genome), a coding sequence for a gRNA under the control of,optionally, an inducible promoter, and a selectable marker.

Once the cells have been transformed with the editing vectors, the cellsare diluted and plated 205 on selective medium 206 to select for cellsthat have both the engine and the editing vectors (e.g., medium withchloramphenicol and carbenicillin) and grown until colonies form 221,again in a process that substantially or largely isolates the cells.Cells are then picked 207 from the colonies 221 and grown overnight in,e.g., first 96-well plate 208 in medium that does not comprise aselective medium. In a next step, the cells from the first 96-well plateare replicated 211 into a second 96-well plate 214 in medium withoutarabinose, resulting in wells with colonies (black wells 220), and wellswithout cells colonies (white wells 222). This process identifies cellswith active gRNAs, as cells with active gRNAs will not survive becausewithout arabinose the λ Red recombineering system is not active torepair the cuts in the genome made by the gRNA. Thus, the wells 222likely denote wells with active gRNAs. The colonies that do grow in thissecond 96-well plate 214 likely have inactive gRNAs, and thus there isno cut to repair and the cells remain viable (denoted by black wells220).

Once the cells with the active gRNAs are identified (wells 222), thesecells are then “cherry-picked” 213 from the first 96-well plate andarrayed into a third 96-well plate 252 to be kept as a cell repositoryor “cell hotel.” All colonies in the third 96-well plate 252 are cellsthat have been identified as likely having active gRNAs 222. With thiscell repository 252, aliquots of these “cherry-picked” cells (e.g., herecells with active gRNAs) can then be arrayed 255 into a fourth 96-wellplate 254 in medium with arabinose. After an initial growth period, thecells in this fourth 96-well plate 254 are subjected to cutting andediting conditions, e.g., increasing the temperature to 42° C. for,e.g., two hours, to induce the pL inducible promoter driving theexpression of the gRNA. Once the cutting and editing processes takeplace, colonies containing cells that have been properly edited can beidentified by monitoring growth of the colonies and selectingslow-growing colonies or by targeted or whole genome sequencing takingcells from plate 254. Once cells with desired edits are identified, thecells with the desired edits can be retrieved from 96-well plate 252(e.g., the cell repository or “cell hotel”). Alternatively, the cellscolonies with the putatively active gRNAs on plate 252 can be pooled 253into a mixed cell culture 256 and either analyzed or subjected to anadditional round of editing.

FIG. 2E depicts yet another exemplary embodiment of an improved protocol270 for performing nucleic acid-guided nuclease genome editing. Theprotocol 270 in FIG. 2D does not entail functional deconvolution of theediting “machinery” of the cells but depicts a protocol forhigh-throughput screening using colony morphology to identify editedcells. Again, in edited cells, cell viability is compromised in theperiod after editing begins. The present method takes advantage of thegrowth lag in colonies of edited cells to identify edited cells. In someembodiments, the colony size of the edited cells is 20% smaller thancolonies of non-edited cells. In some aspects, the colony size of theedited cells is 30%, 40%, 50%, 60%, 70%, 80% or 90% smaller than thecolonies of non-edited cells. In many embodiments, the colony size ofthe edited cells is 30-80% smaller than colonies of non-edited cells,and in some embodiments, the colony size of the edited cells is 40-70%smaller than colonies of non-edited cells.

In FIG. 2E as in FIGS. 2B-2D, a library or collection of editing vectors202 is introduced 203 (e.g., electroporated) into cultured cells 204that comprise a coding sequence for a nuclease, contained 1) on an“engine plasmid” (most often along with a selectable marker) that hasalready been transformed into the cells; 2) integrated into the genomeof the cells being transformed; or 3) the coding sequence for thenuclease may be located on the editing vector. The editing plasmids 202comprise an editing sequence and optionally include a PAM-alteringsequence (e.g., a sequence that disables the PAM at the target site inthe genome), a coding sequence for a gRNA and a selectable marker. Inmany embodiments, a promoter is included in the editing vector backbone,and an editing cassette is inserted 3′ of the promoter where the editingcassette comprises, from 5′ to 3′: a coding sequence for a gRNA, and adonor DNA comprising a desired edit for a target sequence and thePAM-altering sequence (e.g., a CREATE cassette).

At step 271, the transformed cells are diluted and plated (e.g.,substantially or largely isolated) onto selective medium 272 thatselects for both the engine and editing vectors (e.g., medium containingboth chloramphenicol and carbenicillin) and, in bacterial systems,further contains arabinose so as to activate the λ Red recombineeringsystem. Once plated, the cells are grown 273 at 30° C. for 12-16 hoursso that the cells establish colonies, edit, and grow to re-establishcolonies on plate 272. Once colonies appear, there are large 278 andsmall 276 colonies. The colonies with small size 276 are indicative ofan active gRNA and likely to have been edited as the double-strand cutscaused by active editing are largely toxic to the cells, resulting bothin cell death in the edited colonies as well as a lag in growth for theedited cells that do survive but must repair and recover followingediting. The small colonies (edited cells) are cherry picked 277 and arearrayed on a 96-well plate 282 (or several to many 96-well plates).Cells in the 96-well plate 282 can be cultured, and aliquots from this96-well plate 282 can be sequenced and colonies with desired editsidentified. This 96-well plate may be kept as a cell hotel or cellrepository, and once cells that have been properly edited areidentified, one can retrieve the cells with the desired edit from “cellhotel” 96-well plate 282.

Alternatively, small colonies 276 may be picked and pooled 275 foradditional rounds of editing since the edited cells have been selectedin the first round of editing and likely comprise edits from the firstround. Note again, that this method does not provide functionaldeconvolution of the editing machinery; however, the method depicted inFIG. 2E employs both isolation or substantial isolation and cherrypicking and thus enables for a high-throughput diagnostic method toidentify cells based on the colony size morphology that have a highlikelihood of being edited, and once identified, the edited cellpopulation can be enriched. Screening out a large proportion of thecells with non-functional gRNAs allows for identification of editedcells more readily. It has been determined that removing growth ratebias via isolation or substantial isolation improves the observedediting efficiency by up to 2×, 3×, 4× or more over conventionalmethods, and further that cherry-picking colonies using the methodsdescribed herein increases by 1.5×, 1.75×, 2.0×, or 2.5× or more theobserved editing efficiency of isolation. Thus, the combination ofsubstantial isolation and cherry picking improve observed editingefficiency by 8× over conventional methods. Example 9 below providesmaterials and methods for this embodiment.

While the method for screening for edited cells using cell growth as aproxy for editing has been described herein in the context of measuringcolony size of cell colonies on an agar plate, the optical density (OD)of growing cell colonies, such as in a microtiter plate or in a seriesof tubes may be measured instead. Moreover, other cell growth parameterscan be measured in addition to or instead of cell colony size or OD. Forexample, spectroscopy using visible, UV, or near infrared (NIR) lightallows monitoring the concentration of nutrients and/or wastes in thecell culture. Additionally, spectroscopic measurements may be used toquantify multiple chemical species simultaneously. Nonsymmetric chemicalspecies may be quantified by identification of characteristic absorbancefeatures in the NIR. Conversely, symmetric chemical species can bereadily quantified using Raman spectroscopy. Many critical metabolites,such as glucose, glutamine, ammonia, and lactate have distinct spectralfeatures in the IR, such that they may be easily quantified. The amountand frequencies of light absorbed by the sample can be correlated to thetype and concentration of chemical species present in the sample. Eachof these measurement types provides specific advantages. FT-NIR providesthe greatest light penetration depth and so can be used for thickersample so that they provide a higher degree of light scattering.FT-mid-IR (MIR) provides information that is more easily discernible asbeing specific for certain analytes as these wavelengths are closer tothe fundamental IR absorptions. FT-Raman is advantageous when theinterference due to water is to be minimized. Other spectral propertiescan be measured via, e.g., dielectric impedance spectroscopy, visiblyfluorescence, fluorescence polarization, or luminescence. Additionally,sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH,and/or conductivity may be used to assess the rate of cell growth.

FIG. 2F depicts additional detail of the exemplary embodiment shown inFIG. 2E, using a specific example based on experiments performed (seeExample 1, infra). FIG. 2F shows high-throughput screening 290 usingcolony morphology to identify or cherry pick edited cells. As describedabove, in edited cells cell viability is compromised in the period afterediting is induced. The present method takes advantage of the growth lagin colonies of edited cells to identify edited cells. In FIG. 2F,transformed cells are diluted and plated on medium containing arabinose274 and grown for a period of time at, e.g., 30° C. In one embodiment,editing is induced by, e.g., raising the temperature to 42° C. for aperiod of time, then the temperature is lowered to 30° C.; however, inother embodiments editing is not induced and instead is allowed toproceed as the cells grow. Colonies are allowed to grow and both small276 and large 278 colonies result. Colonies from this plate are picked291 and arrayed on a second plate 292 containing selective medium, e.g.,a medium to select for successful editing of galK, resulting in white(versus red) colonies when plated on MacConkey agar supplemented withgalactose as the sole carbon source. Note that picking small colonies293 from the first plate results primarily in edited cells 296 (whitecolonies, shown here as open circles) and—at a much lower frequency—somecells in which the gRNA is inactive 294 (red colonies, shown here asfilled-in circles). Confirmation of colonies in which the gRNA isinactive is shown by picking 295 large colonies 278 from the first plateand plating them on the second plate (colonies 298) where these cellsresult in red colonies when grown on MacConkey agar supplemented withgalactose as the sole carbon source thus confirming an inactive gRNA orother part of the editing machinery. Thus, using small and large colonymorphology as a proxy for edited and non-edited cells, respectively,provides a high throughput and facile screening method for edited cells.Note that the methods depicted in FIGS. 2E and 2F employ both isolationor substantial isolation and cherry-picking strategies.

The exemplary workflows described herein employ the concept ofisolation. Isolation overcomes the growth bias in favor of uneditedcells, thus allowing edited cells “equal billing” with unedited cells.Further, in some embodiments the methods take advantage of atightly-regulated inducible system to screen edited cells fromnon-edited cells (both cells where the gRNA is non-functional, and cellswhere the gRNAs are functional but some component of the nucleicacid-guided nuclease system is not functional). Screening may beperformed using replica plates and identifying “escapees” or cells inwhich the gRNA is non-functional, screening may be performed by takingadvantage of the growth lag of edited cells in comparison to non-editedcells, or enrichment can be performed by using a combination ofisolation or substantial isolation, initial growth, optionally inducingediting followed by normalization. The result of the methods is thateven in nucleic acid-guided nuclease systems where editing is notoptimal (such as in systems where non-canonical PAMs are targeted),there is an increase in the observed editing efficiency; that is, editedcells can be identified even in a large background of unedited cells.

Note that the methods depicted in FIGS. 2B-2F show cell colonyisolation, incubation, and editing on solid medium in cell culturedishes or in 96-well plates. It should be recognized by one of ordinaryskill in the art given the discussion herein that isolation, incubation,and editing can be performed in other formats, such as, e.g., in thesolid wall devices described in relation to FIGS. 3A-3F and 4A-4Z, or asdescribed in U.S. Ser. No. 62/735,365, entitled “Detection of NucleaseEdited Sequences in Automated Modules and Systems”, filed 24 Sep. 2018,and U.S. Ser. No. 62/781,112, entitled “Improved Detection of NucleaseEdited Sequences in Automated Modules and Systems,” filed 18 Dec. 2018,which include descriptions of isolation or substantial isolation byisolating cells on functionalized islands, isolation or substantialisolation within aqueous droplets carried in a hydrophobic carrier fluidor Gel Beads-in-Emulsion (GEMs, see, e.g., 10× Genomics, Pleasanton,Calif.), or isolation or substantial isolation within a polymerizedalginate scaffold (for this embodiment of isolation, also see U.S. Ser.No. 62/769,805, entitled “Improved Detection of Nuclease EditedSequences in Automated Modules and Instruments via Bulk Cell Culture”,filed 20 Nov. 2018).

Exemplary Modules for Editing, Enrichment, and Selection of Edited Cells

The instruments, methods, and modules described herein enable enhancedobserved editing efficiency of nucleic acid-guided nuclease editingmethods as the result of isolation or substantial isolation, incubation,editing, and normalization. The combination of the isolation orsubstantial isolation, incubation, editing and normalization processesovercomes the growth bias in favor of unedited cells—and the fitnesseffects of editing, including differential editing rates—thus allowingall cells “equal billing” with one another. The result of theinstruments, modules, and methods described herein is that even innucleic acid-guided nuclease systems where editing is not optimal (suchas in systems where non-canonical PAMs are targeted), there is anincrease in the observed editing efficiency; that is, edited cells canbe identified even in a large background of unedited cells. Observedediting efficiency can be improved up to 80% or more.

FIG. 3A depicts a solid wall device 350 and a workflow for isolatingcells in microwells in the solid wall device, where in this exemplaryworkflow one or both of the gRNA and nuclease may optionally be underthe control of an inducible promoter. At the top left of the figure (i),there is depicted solid wall device 350 with microwells 352. A section354 of substrate 350 is shown at (ii), also depicting microwells 352. At(iii), a side cross-section of solid wall device 350 is shown, andmicrowells 352 have been loaded, where, in this embodiment, Poisson orsubstantial Poisson loading has taken place; that is, each microwell hasone or no cells, and the likelihood that any one microwell has more thanone cell is low. Note wells 356 have one cell loaded. At (iv), workflow340 is illustrated where substrate 350 having microwells 352 showsmicrowells 356 with one cell per microwell, microwells 357 with no cellsin the microwells, and one microwell 360 with two cells in themicrowell. In step 351, the cells in the microwells are allowed todouble approximately 2-150 times to form clonal colonies (v), thenediting optionally is induced 353 by heating the substrate (e.g., fortemperature-induced editing) or flowing chemicals under or over thesubstrate (e.g., sugars, antibiotics for chemical-induced editing) or bymoving the solid wall device to a different medium, particularly facileif the solid wall device is placed on a membrane which forms the bottomof microwells 352 (membrane not shown).

After optional induction of editing 353, many cells in the colonies ofcells that have been edited die as a result of the double-strand cutscaused by active editing and there is a lag in growth for the editedcells that do survive but must repair and recover following editing(microwells 358), where cells that do not undergo editing thrive(microwells 359) (vi). All cells are allowed to continue grow toestablish colonies and normalize, where the colonies of edited cells inmicrowells 358 catch up in size and/or cell number with the cells inmicrowells 359 that do not undergo editing (vii). Once the cell coloniesare normalized, either pooling 360 of all cells in the microwells cantake place, in which case the cells are enriched for edited cells byeliminating the bias from non-editing cells and fitness effects fromediting; alternatively, colony growth in the microwells is monitoredafter editing, and slow growing colonies (e.g., the cells in microwells358) are identified and selected 361 (e.g., “cherry picked”) resultingin even greater enrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for bacterial growth includes LB, SOC, M9 Minimalmedium, and Magic medium; medium for yeast cell growth includes TPD,YPG, YPAD, and synthetic minimal medium; and medium for mammalian cellgrowth includes MEM, DMEM, IMDM, RPMI, and Hanks. For culture ofadherent cells, cells may be disposed on beads or another type ofscaffold suspended in medium. Most normal mammalian tissue-derivedcells—except those derived from the hematopoietic system—are anchoragedependent and need a surface or cell culture support for normalproliferation. In the rotating growth vial described herein,microcarrier technology is leveraged. Microcarriers of particular usetypically have a diameter of 100-300 μm and have a density slightlygreater than that of the culture medium (thus facilitating an easyseparation of cells and medium for, e.g., medium exchange) yet thedensity must also be sufficiently low to allow complete suspension ofthe carriers at a minimum stirring rate in order to avoid hydrodynamicdamage to the cells. Many different types of microcarriers areavailable, and different microcarriers are optimized for different typesof cells. There are positively charged carriers, such as Cytodex 1(dextran-based, GE Healthcare), DE-52 (cellulose-based, Sigma-AldrichLabware), DE-53 (cellulose-based, Sigma-Aldrich Labware), HLX 11-170(polystyrene-based); collagen or ECM (extracellular matrix)-coatedcarriers, such as Cytodex 3 (dextran-based, GE Healthcare) or HyQ-spherePro-F 102-4 (polystyrene-based, Thermo Scientific); non-chargedcarriers, like HyQspheres P 102-4 (Thermo Scientific); or macroporouscarriers based on gelatin (Cultisphere, Percell Biolytica) or cellulose(Cytopore, GE Healthcare).

The solid wall devices can provide populations of cells with varyingedits and/or percentages of clonality. It has been determined thatflowing medium over the retentate side surface of the SWIIN, e.g., atangential or sheer flow across the top of the perforate member of theSWIIN, will flush off the tops (“muffin tops”, see FIG. 3F) of the cellcolonies over growing the wells of the SWIIN without contaminating thewells containing other cells; i.e., depositing flushed cells in wells.In one embodiment, cells are allowed to grow to a desired state, forexample when some of the colonies—fast-growing colonies, which arelikely to be unedited cells—have over-grown the wells, then the “muffintops” are flushed off. In a next round, the cells again are allowed togrow again to a desired state, for example when some or many more of thecolonies have over-grown the wells, then the “muffin tops” are flushedoff, and additional rounds of cell growth and flushing/collection cancontinue as desired. After a desired number of rounds of cell growth andcollection, 1) the cells can be collected and pooled, 2) certaincollections may be discarded (such as the first collection of cellswhich are more likely to be unedited cells) and the rest of thecollections pooled, or 3) each round of collection may be kept separateand analyzed for clonality, percentage of edited cells, etc. Thisembodiment requires monitoring of cell growth by, e.g., imaging, asdescribed in relation to FIG. 4Y.

FIG. 3B depicts a solid wall device 350 and a workflow for substantiallyisolating cells in microwells in a solid wall device, where in thisworkflow—as in the workflow depicted in FIG. 3A—optionally one or bothof the gRNA and nuclease is under the control of an inducible promoter.At the top left of the figure (i), there is depicted a solid wall device350 with microwells 352. A section 354 of substrate 350 is shown at(ii), also depicting microwells 352. At (iii), a side cross-section ofsolid wall device 350 is shown, and microwells 352 have been loaded,where, in this embodiment, substantial Poisson loading has taken place;that is, one microwell 357 has no cells, and some microwells 376, 378have a few cells. In FIG. 3B, cells with active gRNAs are shown as solidcircles, and cells with inactive gRNAs are shown as open circles. At(iv) workflow 370 is illustrated where substrate 350 having microwells352 shows three microwells 376 with several cells all with active gRNAs,microwell 357 with no cells, and two microwells 378 with some cellshaving active gRNAs and some cells having inactive gRNAs. In step 371,the cells in the microwells are allowed to double approximately 2-150times to form clonal colonies (v), then editing optionally is induced373 by heating the substrate (e.g., for temperature-induced editing) orflowing chemicals under or over the substrate (e.g., sugars, antibioticsfor chemical-induced editing) or by moving the solid wall device to adifferent medium, particularly facile if the solid wall device is placedon a membrane which forms the bottom of microwells 352.

After editing 373, many cells in the colonies of cells that have beenedited die as a result of the double-strand cuts caused by activeediting and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 378),where cells that do not undergo editing thrive (microwells 379) (vi).Thus, in microwells 378 where only cells with active gRNAs reside (cellsdepicted by solid circles), most cells die off; however, in microwells379 containing cells with inactive gRNAs (cells depicted by opencircles), cells continue to grow and are not impacted by active editing.The cells in each microwell (378 and 379) are allowed to grow tocontinue to establish colonies and normalize, where the colonies ofedited cells in microwells 378 catch up in size and/or cell number withthe unedited cells in microwells 379 that do not undergo editing (vii).Note that in this workflow 370, the colonies of cells in the microwellsare not clonal; that is, not all cells in a well arise from a singlecell. Instead, the cell colonies in the well may be mixed colonies,arising in many wells from two to several different cells. Once the cellcolonies are normalized, either pooling 390 of all cells in themicrowells can take place, in which case the cells are enriched foredited cells by eliminating the bias from non-editing cells and fitnesseffects from editing or cells may be flushed from the SWIIN andcollected at various time points; alternatively, colony growth in themicrowells is monitored after editing, and slow growing colonies (e.g.,the cells in microwells 378) are identified and selected 391 (e.g.,“cherry picked”) resulting in even greater enrichment of edited cells.

FIG. 3C is a photograph of one embodiment of a solid wall substratecomprising microwells for isolating cells. As can be seen from thephoto, the solid wall substrate is a perforated disk of metal and isapproximately 2 inches (˜47 mm) in diameter. The perforated disk seen inthis photograph is fabricated 316 stainless steel, where theperforations form the walls of the microwells, and a filter or membraneis used to form the bottom of the microwells. Use of a filter ormembrane (such as a 0.22 μm PVDF Duropore™ woven membrane filter) allowsfor medium and/or nutrients to enter the microwells but prevents thecells from flowing down and out of the microwells. Filter or membranemembers that may be used in the solid wall isolation/incubation/editingand normalization or cherry-picking devices and modules are those thatare solvent resistant, are contamination free during filtration, and areable to retain the types and sizes of cells of interest. For example, inorder to retain small cell types such as bacterial cells, pore sizes canbe as low as 0.10 μm, however for other cell types (such as formammalian cells), the pore sizes can be as high as 10.0 μm. Indeed, thepore sizes useful in the cell concentration device/module includefilters with sizes from 0.10 μm, 0.11 μm, 0.12 μm, 0.13 μm, 0.14 μm,0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm, 0.21 μm, 0.22 μm,0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm,0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm,0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm,0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. The filters may befabricated from any suitable material including cellulose mixed ester(cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

In the photograph shown in FIG. 3C, the perforations are approximately150 μm-200 μm in diameter, resulting in the microwells having a volumeof approximately 2.5 nl, with a total in this embodiment ofapproximately 30,000 microwells. The distance between the microwells isapproximately 279 nm center-to-center. Though here the microwells have avolume of approximately 2.5 nl, the volume of the microwells may be from1 to 25 nl, or preferably from 2 to 10 nl, and even more preferably from2 to 4 nl. The preferred size/volume of the microwells will depend ofcell type (e.g., bacterial, yeast, mammalian). The perforated disk shownhere is made of 316 stainless steel; however other bio-compatible metalsand materials may be used. The solid wall device may be disposable or itmay be reusable. The solid wall device shown in FIG. 3C is round, butcan be of any shape, for example, square, rectangular, oval, etc. (See,e.g., FIGS. 4A-4X.) Round perforated disks are useful if petri dishesare used to supply the solid wall module with nutrients via solid mediumin, e.g., a petri or other cell culture dish. The filters used to formthe bottom of the microwells of the solid wall device include 0.22 μmPVDF Duropore™ woven membrane filters. Further, though a 2-inch (˜47 mm)diameter perforated disk is shown, the perforated disks may be smalleror larger as desired and the configuration of the solid wall module willdepend on how nutrients are supplied to the solid wall module, and howmedia exchange is performed. For example, see the perforated member inthe embodiments of a solid wall module in FIGS. 4A-4T.

FIGS. 3D-3F are photographs of E. coli cells substantially or largelyisolated via Poisson or substantial Poisson distribution in microwellsor perforations in a perforated disk with a membrane bottom at low,medium and high magnification, respectively. FIG. 3D shows digitalgrowth at low magnification where the darker microwells are microwellswhere cells are growing. FIG. 3E is a top view of microwells in aperforated disk where the darker microwells are microwells where cellsare growing. FIG. 3F is a photograph of microwells where the membrane(e.g., the permeable membrane that forms the bottom of the microwells)has been removed, where unpatterned (smooth) microwells are microwellswhere cells are not growing, and microwells with irregularpigment/patterned are microwells where cells are growing, and, in thisphotograph, have filled the microwells in which they are growing. Inthese photographs, a 0.2 μm filter (membrane) was swaged against theperforated disk under high pressure. The perforated disk formed thewalls of the microwells, and the 0.2 μm filter formed the bottom of themicrowells. To load the perforated disk+filter, the E. coli cells werepulled into the microwells using a vacuum (see Example 11 for methods).The perforated disk+filter was then placed on an LB agar platemembrane-side down, and the cells were grown overnight at 30° C., thentwo days at room temperature. The membrane was removed and thebottomless microwells were photographed by light microscopy. Note theease with which different selective media can be used to select forcertain cell phenotypes; that is, one need only transfer the perforateddisk+filter to a different plate or petri dish comprising a desiredselective medium. Generally, the number of cells loaded into asingulation device or singulation assembly ranges from betweenapproximately 0.1× to 2.5× the number of perforations or microwells, orfrom between approximately 0.3× to 2.0× the number of perforations ormicrowells, or from between approximately 0.5× to 1.5× the number ofperforations or microwells.

FIG. 3G shows a scanning electromicrograph of a perforated member. Aswith the perforated disk described in relation to FIGS. 3C-3F,perforated member 301 is fabricated from 316 stainless steel, where theperforations form the walls of microwells, and a filter or membrane (notseen in this FIG. 3G) is used to form the bottom of the microwells. Inthe scanning electromicrograph shown in FIG. 3G the perforations(microwells) are approximately 150 μm-200 μm in diameter, and theperforated member is approximately 125 μm deep, resulting in microwellshaving a volume of approximately 2.5 nl, with a total of approximately200,000 microwells. The distance between the microwells is approximately279 μm center-to-center. Though here the microwells have a volume ofapproximately 2.5 nl, the volume of the microwells may be from 1 to 25nl, or preferably from 2 to 10 nl, and even more preferably from 2 to 4nl. The perforated members used in the embodiments described in relationto FIGS. 4A-4T are approximately 14 cm long and 10 cm (140 mm×100 mm)wide; however, smaller perforated members (such as shown in FIG. 3C) orlarger perforated members may be used depending on the density ofmicrowells in the perforated member and the number of microwells orpartitions required. For example, the larger the plexity (e.g.,complexity) of the library being used to edit a population of cells, alarger number of microwells or partitions is preferred. If, for example,a 10,000-plex library is used to edit a population of cells, aperforated member with 200,000 microwells or partitions would be morethan adequate; however, if a 50,000-plex library is used to edit apopulation of cells, a perforated member (or two or more members in acompound SWIIN) with a total of 400,000 or more microwells or partitionsmay be preferred. The number of microwells in a single SWIIN module(e.g., not a compound SWIIN) may range from 20,000 to 500,000microwells, or from 30,000 to 450,000 microwells, or from 50,000 to400,000 microwells or from 100,000 to 300,000 microwells. For an evengreater range of well number, compound SWIIN devices may be employed(see, e.g., FIGS. 4CC and 4DD of U.S. Ser. No. 16/399,988, filed 30 Apr.2019).

Generally the number of cells loaded into a singulation device orsingulation assembly ranges from between approximately 0.1× to 2.5× thenumber of perforations or microwells, or from between approximately 0.3×to 2.0× the number of perforations or microwells, or from betweenapproximately 0.5× to 1.5× the number of perforations or microwells;thus, the number of cells loaded onto a perforated member comprisingapproximately 200,000 perforations would range from about 20,000 cellsto about 500,000 cells, or from about 60,000 cells to about 400,000cells, or from about 100,000 cells to about 300,000 cells. The preferredsize/volume of the microwells will depend on the cell type (e.g.,archaeal, bacterial, yeast, non-mammalian eukaryotic, and/or mammalianbeing edited). The perforated member shown here is made of 316 stainlesssteel; however other bio-compatible metals and materials may be used,such as titanium, cobalt-based alloys, and ceramics. The SWIIN may bedisposable or it may be reusable. If reused, the SWIIN may be heated to55° C. or greater to sterilize the SWIIN alternatively, antibioticsmaybe flushed through the SWIIN.

Like FIG. 3G, FIGS. 3H-3J are scanning electromicrographs of a portionof a perforated member 301 (FIG. 3G), a close up of one microwell 302without (FIG. 3H) and with (FIGS. 3I and 3J) a filter membrane formingthe bottom of the microwell 302. FIG. 3G shows perforated member 301 and30 or so microwells 302. FIG. 3H also shows perforated member 301 and asingle microwell 302, where this microwell is approximately 172 μm indiameter. FIG. 3I shows perforated member 301 and approximately 8microwells 302, each of which has a portion of a filter or membrane 303forming the bottom of the microwell 302. FIG. 3J is a highermagnification micrograph of one of the microwells 302 from theperforated member 301 shown in FIG. 3I.

As described above in relation to FIGS. 3C-3F, use of a filter ormembrane (such as a 0.22 μm PVDF Duropore™ woven membrane filter) allowsfor medium and/or nutrients to enter the microwells but prevents cellsfrom flowing down and out of the microwells. Filter or membrane membersthat may be used to form the bottom of the microwells of a perforatedmember in a solid wall isolation or substantialisolation/incubation/editing/normalization or cherry picking module arethose that are solvent resistant, are contamination free duringfiltration, do not tear under pressures required to exchange media andload cells, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 5.0 μm or larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber. The perforatedmember 301 and filter 303 are swaged together; that is, the perforatedmember 301 and filter 303 are pressed together under high pressure(e.g., 20 kpsi). In some embodiments, the holes or partitions in theperforated member are etched with a slight taper and the filter materialis pressed into the opening then relaxed to form an effective swage,allowing for an adhesive-free coupling. As an alternative, an adhesivecould be employed.

FIGS. 4A through 4Z depict various components of different embodimentsand components of a solid wall isolation or substantial isolation,incubation, editing and either normalization or cherry picking module(“solid wall isolation/incubation/normalization module” or “SWIIN”)suitable for isolating (or substantially isolating) cells of all types,growing cells for an initial, e.g., 2-150 rounds of cell division,optionally inducing editing, and either normalizing or cherry pickingthe resulting cell colonies. The SWIIN modules presented may bestand-alone devices, or, often, one module in an automated multi-modulecell processing instrument.

FIG. 4A depicts an embodiment of a singulation assembly 420 a from a topperspective view, which presents the “retentate side” of singulationassembly 420 a. In the singulation assembly 420 a embodiment seen inFIG. 4A, retentate member 404 in FIGS. 4A-4C has two distributionchannels 405 disposed lengthwise on either side (left-right) ofretentate member 404; however, it should be understood by one ofordinary skill in the art given the present disclosure that instead oftwo distribution channels, a single distribution channel could bedisposed, e.g., lengthwise down the middle of retentate member 404 or inanother configuration three distribution channels may be disposedlengthwise on either side and down the middle of retentate member 404.Retentate member 404 of FIGS. 4A-4C comprises a generally smooth uppersurface; two distribution channels 405 which traverse retentate member404 from its top surface to its bottom surface and for most of thelength of retentate member 404; and ridges 406 a, which are disposed onthe bottom surface of retentate member 404 and traverse the bottom ofretentate member 404 from side-to-side, left-to-right (in thisembodiment, there are approximately 26 retentate member ridges 406 a).Flow directors 406 are formed between ridges 406 a on retentate member404. In addition, retentate member 404 has two ports 407, which allowfor cells and medium to be introduced into (and removed from)singulation assembly 420 a. There are also distribution channel covers413, which cover the two distribution channels 405 and provide the topsurface and seal of distribution channels 405.

As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. Flow directors 406 are formed between ridges 406 a. Inaddition, retentate member 404 has a single port 407, which allows forcells to be introduced into singulation assembly 420 a; and there isalso a distribution channel cover 413, which covers the singledistribution channel 405 in retentate member 404 and provides a seal fordistribution channel 405. In this embodiment, distribution channel 405is approximately 150 mm long and 1 mm wide; retentate member ridges 406a are approximately 0.5 mm in height and 80 mm in length; and retentatemember flow directors 406 are approximately 5 mm across. The volume offluid in the singulation assembly ranges from 3 mL to 100 mL, or from 5mL to 60 mL, or from 10 mL to 40 mL (note this is for a 200K perforationsingulation assembly).

Retentate and permeate members 404 and 408, respectively, in theembodiments exemplified in FIG. 4A-4N are transparent, and areapproximately 200 mm long, 130 mm wide, and 4 mm thick, though in otherembodiments, the retentate and permeate members can be from 75 mm to 350mm in length, or from 100 mm to 300 mm in length, or from 150 mm to 250mm in length; from 50 mm to 250 mm in width, or from 75 mm to 200 mm inwidth, or from 100 mm to 150 mm in width; and from 2 mm to 15 mm inthickness, or from 4 mm to 10 mm in thickness, or from 5 mm to 8 mm inthickness. In the embodiments depicted in FIGS. 4A-4N, the retentate(and permeate) members are fabricated from PMMA (poly(methylmethacrylate); however, other materials may be used, includingpolycarbonate, cyclic olefin co-polymer (COC), glass, polyvinylchloride, polyethylene, polyamide, polypropylene, polysulfone,polyurethane, and co-polymers of these and other polymers.

In addition to retentate member 404, also seen in FIG. 4A are fastenerholes 412, a center “sandwich” layer comprising a gasket 416 surroundingperforated member 401 swaged with (and positioned above) filter ormembrane 403 (the individual components are not seen in this FIG. 4A).The bottom layer of singulation assembly 420 a seen in FIG. 4A is formedby a permeate member 408. Permeate member 408, like retentate member404, comprises one or more (as seen in FIG. 4B, there are two) permeatemember distribution channels, a multiplicity of ridges, and flowdirectors, and one or more ports (none of which are shown in FIG. 4A butsee FIG. 4B). The singulation assembly 420 a and SWIIN modules 400comprising the singulation assembly 420 a are fabricated from materialthat withstand temperature of 4° C. to 60° C. Heating and cooling of theSWIIN modules may be provided by a Peltier device or thermoelectriccooler or by reverse Rankine vapor-compression refrigeration orabsorption heat pumps.

In the solid wall isolation or substantial isolation, incubation,editing and either normalization or cherry-picking modules (“solid wallisolation/incubation/normalization module” or “SWIIN”) described inFIGS. 4A-4N, cells and medium (at a dilution appropriate for Poisson orsubstantial Poisson distribution of the cells in the microwells of theperforated member) are flowed into distribution channels 405 from thetwo ports 407 in retentate member 404, and the cells settle in themicrowells, again, resulting in a Poisson or substantial Poissondistribution of the cells in the microwells. The cells are retained inthe microwells of perforated member 401 as the cells cannot travelthrough filter 403. An appropriate medium is introduced into singulationassembly 420 a through permeate member 408. The medium flows upwardthrough filter 403 to nourish the cells. Thus, in operation, the cellsare deposited into the microwells, are grown for an initial, e.g., 2-100doublings, editing optionally is induced by, e.g., raising thetemperature of the SWIIN to 42° C. to induce a temperature induciblepromoter or by removing growth medium from the permeate member andreplacing the growth medium with a medium comprising a chemicalcomponent that induces an inducible promoter. Once editing has beenallowed to take place, the temperature of the SWIIN may be decreased, orthe inducing medium may be removed and replaced with fresh mediumlacking the chemical component thereby de-activating the induciblepromoter. The cells then are allowed to continue to grow in the SWIINuntil the growth of the cell colonies in the microwells is normalized.Once the colonies are normalized, the colonies are flushed from themicrowells (by applying liquid or air pressure to the permeate memberchannels and flow directors and thus to the filter) and pooled;alternatively, the growth of the cell colonies in the microwells ismonitored, and slow-growing colonies are directly selected by, e.g.,pooling the cells from the slow-growing colonies.

Colony growth in the singulation assembly (and thus in the SWIIN module)can be monitored by automated devices such as those sold by JoVE(ScanLag™ system, Cambridge, Mass.) (also see Levin-Reisman, et al.,Nature Methods, 7:737-39 (2010)). Cell growth for, e.g., mammalian cellsmay be monitored by, e.g., the growth monitor sold by IncuCyte (AnnArbor, Mich.) (see also, Choudhry, PLos One, 11(2):e0148469 (2016)).Further, automated colony pickers may be employed, such as those soldby, e.g., TECAN (Pickolo™ system, Mannedorf, Switzerland); Hudson Inc.(RapidPick™, Springfield, N.J.); Molecular Devices (QPix 400™ system,San Jose, Calif.); and Singer Instruments (PIXL™ system, Somerset, UK).

FIG. 4B depicts the embodiment of the singulation assembly 420 a in FIG.4A from a bottom perspective view, which presents the bottom of thepermeate member 408 of singulation assembly 420 a. Permeate member 408comprises a generally smooth lower surface (which in FIG. 4B, becausesingulation assembly 420 a is viewed from the bottom, the lower surfaceis toward the top of FIG. 4B); two distribution channels (not shown)covered by distribution channel covers 414 (here, located on either side(left-right) of permeate member 408); ridges 410 a disposed on the topsurface of permeate member 408 (where the top surface of the permeatemember 408 in FIG. 4B is facing down) and traverse the top of permeatemember 408 from side-to-side (in this embodiment, there areapproximately 26 ridges 410 a); and flow directors 410 that are formedbetween ridges 410 a. In addition, permeate member 408 has two ports411, which deliver fluids to (and remove fluids from) the distributionchannels (not seen here as they are covered by distribution channelcovers 414) and then to flow directors 410.

In addition to permeate member 404, also seen in FIG. 4B are fastenerholes 412, a center “sandwich” layer comprising a gasket 416 surroundinga perforated member 401 swaged with and positioned above a filter ormembrane 403 (the individual components are not seen in this FIG. 4B),and the bottom-most layer of singulation assembly 420 a seen in FIG. 4Bis retentate member 404. Gasket 416 is made from rubber, silicone,nitrile rubber, polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other readily compressible material. Itshould be noted that in the embodiments depicted in FIGS. 4A-4H,fasteners are exemplified; however, other means can be used to secureretentate member 408, gasket 416, perforated member 401, filter 403, andpermeate member 408, such as adhesives, ultrasonic welding or bonding,solvent bonding, mated fittings, or a combination of adhesives, welding,solvent bonding, and mated fittings; and other such fasteners andcouplings. Note in the embodiment exemplified in FIGS. 4Q-4X, ultrasonicwelding is used.

FIG. 4C depicts the embodiment of the singulation assembly 420 a ofFIGS. 4A and 4B from a side exploded perspective view. From the top ofFIG. 4C is seen retentate member 404 comprising two distributionchannels 405 located on either side (left-right) of retentate member404, both of which traverse retentate member 404 from its top surface toits bottom surface and for most of the length of retentate member 404;ridges 406 a, which are disposed on the bottom surface of retentatemember 404 and traverse the bottom surface of retentate member 404 fromside-to-side (in this embodiment, there are approximately 26 ridges 406a); and flow directors 406 that are formed between ridges 406 a. Inaddition, retentate member 404 has two ports 407, which allow for cellsand other fluids to be introduced into (and removed from) singulationassembly 420 a through distribution channels 405. There are also twodistribution channel covers 413, which are configured to fit into, coverand seal the two distribution channels 405.

Gasket 416, perforated member 401 and filter 403 can be seen moreclearly in this exploded view of singulation assembly 420 a, whereperforated member 401 and filter 403 are of very similar size and gasket416 is configured to surround perforated member 401 and filter 403, tosecure perforated member 401 and filter 403, and to provide a leak-proofseal between retentate member 404 and permeate member 408. In FIG. 4C,permeate member 408 is seen from the top down where permeate member 408comprises two distribution channels 409 located lengthwise on eitherside (left-right) of permeate member 408, both of which traversepermeate member 408 from its bottom surface to its top surface and formost of the length of permeate member 408. Also seen are ridges 410 a,which here are disposed on the top surface of permeate member 408 andtraverse the top of permeate member 408 from side-to-side, and flowdirectors 410 that are formed between ridges 410 a. In addition,permeate member 408 has two ports 411, seen in this FIG. 4C as ports onretentate member 404, which when singulation assembly 420 a is assembledare fluidically connected to distribution channels 409 on permeatemember 408, which are connected to flow distributors 410, and thus tofilter 403 disposed upon permeate member 408. Also seen are distributionchannel covers 414 that are separate from singulation assembly 420 a inthis exploded view and not inserted into permeate member 408 to coverdistribution channels 409. Also seen in FIG. 4C are holes for fasteners412 (fasteners not shown), although again, other means aside fromfasteners may be used for securing the components of singulationassembly 420 a.

FIG. 4D depicts another embodiment of a singulation assembly, 420 b,from a top perspective view, which presents the “retentate side” ofsingulation assembly 420 b. The singulation assembly 420 b embodimentseen in FIG. 4D differs from singulation assembly 420 b seen in FIGS.4A-4C in that the configuration of the distribution channels 405 inretentate member 404 (and distribution channels 409 in permeate member408) in FIGS. 4D-4G is different. The distribution channels 405 (anddistribution channels 409) in FIGS. 4D-4G are branched; that is, insteadof a port 407 delivering fluids directly into a distribution channel 405at the point where port 407 intersects directly with distributionchannel 405, in the embodiment of the singulation assembly shown inFIGS. 4D-4G, fluids flow into ports 407, then into branched distributionchannels 405 on either side (left-right) of retentate member 407. Thebranched distribution channels in this embodiment have a first conduitwhich terminates approximately half-way down the length of retentatemember 404, where the first conduit then branches into two secondaryconduits, the two secondary conduits branch into four tertiary conduits,and these tertiary conduits deliver fluids to a final conduit thattravels the length of the retentate member, evenly distributingdelivering fluids to the flow directors 406.

Note that any configuration of distribution channels (e.g., distributionchannels 405 on retentate member 404 and distribution channels 409 onpermeate member 408) may be used on retentate member 404 or permeatemember 408, as long as the distribution channels adequately distributefluids to flow directors 406 or flow directors 410. As in FIGS. 4A-4C,retentate member 404 of FIGS. 4D-4G comprises a generally smooth uppersurface; two distribution channels 405 one of the left and one on theright of retentate member 404 which traverse retentate member 404 fromits top surface to its bottom surface and are branched thus distributingfluid the length of retentate member 404.

Retentate member 404 also comprises ridges 406 a, which are disposed onthe bottom surface of retentate member 404 and traverse the bottom ofretentate member 404 from side-to-side. In addition—and as in theprevious embodiment—there are flow directors 406 that are formed betweenridges 406 a on retentate member 404; and two ports 407, which introduceand distribute cells and medium into (and remove cells and medium from)retentate member 404 of singulation assembly 420 b. Also seen aredistribution channel covers 413, which cover the two brancheddistribution channels 405 on retentate member 404 and actually providethe top surface and sealing of branched distribution channels 405. Notethat the branching for distribution channels 405 is a part of retentatemember 404 in this embodiment; however, retentate member 404 may notcomprise the branches and the branching may be featured on distributionchannel covers 413, which are mated to retentate member 404.

In addition to retentate member 404, also seen in FIG. 4D are fastenerholes 412, a center “sandwich” layer comprising a gasket 416 surroundingperforated member 401 swaged with and positioned above filter ormembrane 403. The bottom layer of singulation assembly 420 b seen inFIG. 4D is formed by permeate member 408. Permeate member 408, likeretentate member 404, comprises one or more (as seen in FIG. 4E, thereare two) distribution channels (here, they are branched), ridges, flowdirectors, and one or more ports (none of which are shown in FIG. 4D butsee FIG. 4E).

FIG. 4E depicts the embodiment of the singulation assembly 420 b in FIG.4D from a bottom perspective view, which presents the bottom of thepermeate member 408 of singulation assembly 420 b at the top of thisFIG. 4E. Permeate member 408 comprises a generally smooth lower surface(which in FIG. 4E, because singulation assembly 420 b is viewed from thebottom, the lower surface is toward the top of FIG. 4E); two brancheddistribution channels 409 covered by distribution channel covers 414;ridges 410 a disposed on the top surface of permeate member 408 (wherethe top surface of the permeate member 408 in FIG. 4E is facing down)and traverse the top surface of permeate member 408 from side-to-side;and flow directors 410 that are formed between ridges 410 a. Inaddition, permeate member 408 has two ports 411, which deliver fluids tothe branched distribution channels 409 and to flow directors 410. Aswith retentate member 404 and distribution channel covers 413, thebranching for distribution channels 409 in permeate member 408 is a partof permeate member 408; however, permeate member 408 may not comprisethe branches and the branching may instead be featured on distributionchannel covers 414, which are mated to permeate member 408.

In addition to permeate member 404, also seen in FIG. 4E are fastenerholes 412, a center “sandwich” layer comprising a gasket 416 surroundinga perforated member 401 disposed above a filter or membrane 403 (theindividual components are not seen in this FIG. 4E), and bottom-mostlayer of singulation assembly 420 b seen in FIG. 4E is the retentatemember 404.

FIG. 4F depicts the embodiment of the singulation assembly 420 b ofFIGS. 4D and 4E from a side exploded perspective view. From the top ofFIG. 4F is seen retentate member 404 comprising two brancheddistribution channels 405 located on either side (left-right) ofretentate member 404, both of which traverse retentate member 404 fromits top surface to its bottom surface where the branches traverse mostof the length of retentate member 404; ridges 406 a disposed on thebottom surface of retentate member 404 and traverse the bottom surfaceof retentate member 404 from side-to-side; and flow directors 406 thatare formed between ridges 406 a. In addition, retentate member 404 hastwo ports 407, which are fluidically coupled to branched distributionchannels 405 and flow directors 406 and are configured to introducecells and other fluids into (and remover cells and other fluids from)singulation assembly 420 b. There are also distribution channel covers413, which are configured to fit into, cover and seal the two brancheddistribution channels 405.

Gasket 416, perforated member 401 and filter 403 can be seen clearly inthis exploded view of singulation assembly 420 b, where perforatedmember 401 and filter 403 are of very similar size and gasket 416 isconfigured to surround perforated member 401 and filter 403 and toprovide a leak-proof seal between retentate member 404 and permeatemember 408. In FIG. 4F, permeate member 408 is seen from the top downwhere permeate member 408 comprises two branched distribution channels409 located lengthwise on either side (left-right) of permeate member408, both of which traverse permeate member 408 from its bottom surfaceto its top surface and provide branched conduits for most of the lengthof permeate member 408. Permeate member 408 further comprises ridges 410a, which here are disposed on the top surface of permeate member 408 andtraverse the top of permeate member 408 from side-to-side and flowdirectors 410 that are formed between permeate member ridges 410 a. Inaddition, permeate member 408 has two ports 411 (only one port 411 canbe seen in FIG. 4F). Also seen are distribution channel covers 414 thatare separate from singulation assembly 420 b in this exploded view andnot inserted to cover branched distribution channels 409. Also seen inFIG. 4F are fastener holes 412 and fasteners 412 a, although again,other means aside from fasteners may be used for securing the componentsof singulation assembly 420 b.

FIG. 4G is a close-up top view from the top of retentate member 404 asshown in FIGS. 4D and 4F. Seen are ridges 406 a, which are disposed onthe bottom surface of retentate member 404 and traverse the bottomsurface of retentate member 404 from side-to-side, and flow directors406 that are formed between ridges 406 a. Additionally seen isdistribution channel 405 comprising flow director holes 415 configuredto distribute fluid into flow directors 406, where distribution channel405 is covered by distribution channel cover 413.

FIG. 4H is a close-up cross-sectional view of retentate member 404, withridges 406 a and flow directors 406 that are formed between ridges 406a. Also seen is permeate member 408, with ridges 410 a and flowdirectors 410 that are formed between permeate member ridges 410 a.Interposed between retentate member 404 and permeate member 408 aregasket 416, perforated member 401, and filter 403. Note that ridges 406a on retentate member 404 and ridges 410 a are coincident with oneanother, separated only by perforated member 401 and filter 403. Asstated previously, ridges 406 a and ridges 410 a provide support toperforated member 401 and filter 403 and reduce the likelihood thatfilter 403 will tear during, e.g., cell loading or medium exchange.

FIGS. 4I-4M depict different views of one embodiment of a solid wallisolation or substantial isolation, incubation, editing andnormalization (SWIIN) module 400. FIG. 4I presents a side perspectiveview of SWIIN module 400. SWIIN module 400 comprises, e.g., one of theexemplary singulation assemblies seen in FIGS. 4A-4C and 4D-4F which arehoused in a SWIIN cover 440 and are one part of the SWIIN module.Various components of SWIIN cover 440 include reservoir cover 442, grip441, windows 444 (shown are six windows), feet 443, and barcode (orother identifying information) 445. Note that in the embodimentsdepicted in FIGS. 4I and 4J the windows are round; however, it should berecognized by one of ordinary skill in the art given the presentdisclosure that the windows can be any shape. Typically, it is desirablefor 30% or more of the retentate member be available for viewing, or 50%more, or 60% more, or 70%, 80% or 90% more of the retentate member beavailable for viewing to get a reasonable sub-sample statistic on cellloading. Also seen is reservoir assembly cover 430 a, which in thisembodiment is not molded with SWIIN cover 440 but resides within thereservoir cover portion 442 of SWIIN cover 440. Reservoir assembly cover430 a comprises four reservoir access apertures (432 a, 432 b, 432 c,and 432 d) and four pneumatic access apertures (433 a, 433 b, 433 c, and433 d). Pneumatic access apertures 433 a, 433 b, 433 c, and 433 d inmost embodiments include filters to prevent contamination.

Windows 444 may be used to monitor cell loading and/or cell growth viacamera (e.g., video camera). For example, a video camera may be used tomonitor cell growth by, e.g., density change measurements based on animage of an empty well, with phase contrast, or if, e.g., a chromogenicmarker, such as a chromogenic protein, is used to add a distinguishablecolor to the cells. Chromogenic markers such as blitzen blue, dreidelteal, virginia violet, vixen purple, prancer purple, tinsel purple,maccabee purple, donner magenta, cupid pink, seraphina pink, scroogeorange, and leor orange (the Chromogenic Protein Paintbox, all availablefrom ATUM (Newark, Calif.)) obviate the need to use fluorescence,although fluorescent cell markers, fluorescent proteins, andchemiluminescent cell markers may also be used.

FIG. 4J is a top-down view of SWIIN module 400, showing SWIIN cover 440and various components thereof including reservoir cover 442, grip 441,windows 444 (shown are six windows), feet 443, and barcode (or otheridentifying information) 445. Also seen is reservoir assembly cover 430a residing within the reservoir cover portion 442 of SWIIN cover 440,where reservoir assembly cover 430 a comprises four reservoir accessapertures (432 a, 432 b, 432 c, and 432 d) and four pneumatic accessapertures (433 a, 433 b, 433 c, and 433 d).

FIG. 4K is a side view of SWIIN module 400, depicting SWIIN cover 440,reservoir cover 442, grip 441, and feet 443. FIG. 4L is a view from thebarcode “end” of SWIIN module 400 toward the reservoir “end” of SWIINmodule 400. Seen are SWIIN cover 400, reservoir cover 442, grip 441, andfeet 443. FIG. 4M is a bottom perspective view of SWIIN module 400,showing SWIIN cover 440, reservoir cover 442, feet 443, as well asdistribution channel covers 414 disposed on the bottom of permeatemember 408.

FIGS. 4N and 4O depict views of a reservoir assembly 430. In FIG. 4N,reservoir assembly 430 is disposed upon a singulation assembly 420 acomprising retentate member 404; distribution channel covers 413; agasket, perforated member, and filter assembly (not shown clearly), anda permeate member (also not shown clearly) Reservoir assembly 430comprises reservoir assembly cover 430 a, where reservoir assembly cover430 a comprises four reservoir access apertures (432 a, 432 b, 432 c,and 432 d) and four pneumatic access apertures (433 a, 433 b, 433 c, and433 d).

FIG. 4O is a top perspective view of a cross section of reservoirassembly 430 taken through the “reservoir” end of SWIIN cover 440. Seenare feet 443 of SWIIN cover 440. Reservoirs 431 a, 431 b, 431 c, and 431d are seen, as well as reservoir/channel ports 434 a, 434 b, 434 c, and434 d. Reservoirs 431 b and 431 c are fluidically coupled to conduitsthat either are fluidically coupled to distribution channels on theretentate member or distribution channels on the permeate member, andreservoirs 431 a and 431 d are fluidically coupled to conduits thateither are fluidically coupled to distribution channels on the retentatemember or distribution channels on the permeate member; that is ifreservoirs 431 b and 431 c are fluidically coupled to conduits that arecoupled to distribution channels on the retentate member, thenreservoirs 431 a and 431 d are fluidically coupled to conduits that arecoupled to distribution channels on the permeate member. However, anyreservoir may be configured to deliver fluids to and remove fluids fromeither retentate member 404 or permeate member 408. Reservoirs 431 a,431 b, 431 c, and 431 d typically have a volume of from 5.0 to 100 mL,or from 7.5 to 60 mL, or from 10 to 40 mL (these volumes are for the200K singulation assembly). Note that reservoirs 431 a, 431 b, 431 c,and 431 d are funnel-shaped in the portion of the reservoir leading toreservoir/channel ports 434 a, 434 b, 434 c, and 434 d and aid indelivery of fluids from reservoirs 431 a, 431 b, 431 c, and 431 d.

FIG. 4P is an exemplary pneumatic block diagram suitable for the SWIINmodule depicted in FIGS. 4I-4M and, e.g., utilizing the singulationassemblies described in relation to FIGS. 4A-4F. Note that there are twosolenoid valves (SV) for each reservoir—two retentate reservoirs (RR1and RR2) and two permeate reservoirs (PR1 and PR2)—where one of thevalves is for pneumatic actuation, and one of the valves serves to blockthe line. Note there is a flow meter for each pair of solenoid valves.Also, in this embodiment there is a single manifold serving allreservoirs; however, other embodiments may employ two manifolds, withPR1 and RR1 served by one manifold and PR2 and RR2 served by anothermanifold, or each reservoir (PR1, RR1, PR2, RR2) served by a separatemanifold. Tables 1 and 2 provide the valve status, pressures, andreservoir volumes for each step of the initial fluid flow, cell loading,growth, editing, and cell collection processes for the pneumatic blockdiagram in FIG. 4P.

FIG. 4Q depicts yet another embodiment of a SWIIN module 450 from anexploded top perspective view. The SWIIN module embodiment described inrelation to FIGS. 4Q-4Z provides advantages over thepreviously-described SWIIN modules. For example, the positioning of thereservoirs and reservoir ports below the retentate and permeateserpentine channels minimizes instantaneous flow of fluid in thereservoirs through the reservoir ports and into channels that connectthe reservoir ports to the retentate and permeate channels. Instead,flow is controlled by the application of pressure (positive or negative)and an appropriate time chosen by the user. Additionally, unlike theprevious SWIIN embodiments described herein, SWIIN module 450 does nothave a “singulation assembly” comprising the retentate and permeatemembers and a gasket surrounding the perforated member and filtersandwiched between the retentate and permeate members that is separatefrom a SWIIN cover and reservoir assembly; instead, in SWIIN module 450the retentate member is formed on the bottom of a top of a SWIIN modulecomponent and the permeate member is formed on the top of the bottom ofa SWIIN module component Eliminating a “singulation assembly”, SWIINcover, SWIIN grip, etc., vastly simplifies manufacture and assembly ofthe SWIIN module and decreases costs. In addition, a SWIIN assemblycomprising the SWIIN module 450 comprises features to managecondensation, which allows for improved imaging of the wells. Thesefeatures are described infra.

Thus, the SWIIN module 450 in FIG. 4Q comprises from the top down, areservoir gasket or cover 458, a retentate member 404 (where a retentateflow channel cannot be seen in this FIG. 4Q), a perforated member 401swaged with a filter (filter not seen in FIG. 4Q), a permeate member 408comprising integrated reservoirs (permeate reservoirs 452 and retentatereservoirs 454), and two reservoir seals 462, which seal the bottom ofpermeate reservoirs 452 and retentate reservoirs 454. A permeate channelcan be seen disposed on the top of permeate member 408, defined by araised portion 476 of serpentine channel 460 a, and ultrasonic tabs 464can be seen disposed on the top of permeate member 408 as well. Theperforations that form the wells on perforated member 401 are not seenin this FIG. 4Q; however, through-holes 466 to accommodate theultrasonic tabs 464 are seen. In addition, supports 470 are disposed ateither end of SWIIN module 450 to support SWIIN module 450 and toelevate permeate member 408 and retentate member 404 above reservoirs452 and 454 to minimize bubbles or air entering the fluid path from thepermeate reservoir to serpentine channel 460 a or the fluid path fromthe retentate reservoir to serpentine channel 460 b (neither fluid pathis seen in this FIG. 4Q, but see FIG. 4X).

In this FIG. 4Q, it can be seen that the serpentine channel 460 a thatis disposed on the top of permeate member 408 traverses permeate member408 for most of the length of permeate member 408 except for the portionof permeate member 408 that comprises permeate reservoirs 452 andretentate reservoirs 454 and for most of the width of permeate member408. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member is similarto the perforated member described in relation to the SWIIN singulationassembly in FIGS. 4A-4H with the exception that the perforated member inthis embodiment includes through-holes to accommodate ultrasonic tabsdisposed on the permeate member. Thus, in this embodiment the perforatedmember is fabricated from 316 stainless steel, and the perforations formthe walls of microwells while a filter or membrane is used to form thebottom of the microwells. Typically, the perforations (microwells) areapproximately 150 μm-200 μm in diameter, and the perforated member isapproximately 125 μm deep, resulting in microwells having a volume ofapproximately 2.5 nl, with a total of approximately 200,000 microwells.The distance between the microwells is approximately 279 μmcenter-to-center. Though here the microwells have a volume ofapproximately 2.5 nl, the volume of the microwells may be from 1 to 25nl, or preferably from 2 to 10 nl, and even more preferably from 2 to 4nl. As for the filter or membrane, like the filter described previously,filters appropriate for use are solvent resistant, contamination freeduring filtration, and are able to retain the types and sizes of cellsof interest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.10 μm, however for othercell types (e.g., such as for mammalian cells), the pore sizes can be ashigh as 10.0 μm-20.0 μm or more. Indeed, the pore sizes useful in thecell concentration device/module include filters with sizes from 0.10μm, 0.11 μm, 0.12 μm, 0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18μm, 0.19 μm, 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50μm and larger. The filters may be fabricated from any suitable materialincluding cellulose mixed ester (cellulose nitrate and acetate) (CME),polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone(PES), polytetrafluoroethylene (PTFE), nylon, or glass fiber.

FIG. 4R is a top-down view of permeate member 408, showing serpentinechannel 460 a (the portion of the serpentine channel disposed inpermeate member 408) defined by raised portion 476 of serpentine channel460 a, permeate reservoirs 452, retentate reservoirs 454, reservoirports 456 (two of the four of which are labeled), ultrasonic tabs 464disposed at each end of permeate member 408 and on the raised portion476 of serpentine channel 460 a of permeate member 408, two permeateports 411, and two retentate ports 407 are also seen.

FIG. 4S is a bottom-up view of retentate member 404, showing serpentinechannel 460 b (the portion of the serpentine channel disposed inretentate member 408) defined by the raised portion 476 of theserpentine channel 460 b. Also seen is an integrated reservoir cover 478for the permeate and retentate reservoirs that mate with permeatereservoirs 452 and retentate reservoirs 454 on the permeate member. Theintegrated reservoir cover 478 comprises reservoir access apertures 432a, 432 b, 432 c, and 432 d, as well as pneumatic ports 433 a, 433 b, 433c and 433 d. As with previous embodiments, the serpentine channel 460 aof permeate member 408 and the serpentine channel 460 b of retentatemember 404 mate to form the top (retentate member) and bottom (permeatemember) of a mated serpentine channel. The footprint length of theserpentine channel structure is from, e.g., from 80 mm to 500 mm, from100 mm to 400 mm, or from 150 mm to 250 mm. In some aspects, the entirefootprint width of the channel structure is from 50 mm to 200 mm, from75 mm to 175 mm, or from 100 mm to 150 mm.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mmin hydraulic radius.

As in previous embodiments, disposed between serpentine channels 460 aand 460 b is perforated member 401 (adjacent retentate member 404) andfilter 403 (adjacent permeate member 408), where filter 403 is swagedwith perforated member 401. Serpentine channels 460 a and 460 b can haveapproximately the same volume or a different volume. For example, each“side” or portion 460 a, 460 b of the serpentine channel may have avolume of, e.g., 2 mL, or serpentine channel 460 a of permeate member408 may have a volume of 2 mL, and the serpentine channel 460 b ofretentate member 404 may have a volume of, e.g., 3 mL. The volume offluid in the serpentine channel may range from about 2 mL to about 80mL, or about 4 mL to 60 mL, or from 5 mL to 40 mL, or from 6 mL to 20 mL(note these volumes apply to a SWIIN module comprising a, e.g., 50-500Kperforation member). The volume of the reservoirs may range from 5 mL to50 mL, or from 7 mL to 40 mL, or from 8 mL to 30 mL or from 10 mL to 20mL, and the volumes of all reservoirs may be the same or the volumes ofthe reservoirs may differ (e.g., the volume of the permeate reservoirsis greater than that of the retentate reservoirs).

The serpentine channel portions 460 a and 460 b of the permeate member408 and retentate member 404, respectively, are approximately 200 mmlong, 130 mm wide, and 4 mm thick, though in other embodiments, theretentate and permeate members can be from 75 mm to 400 mm in length, orfrom 100 mm to 300 mm in length, or from 150 mm to 250 mm in length;from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness. As inpreviously described embodiments the retentate (and permeate) membersmay be fabricated from PMMA (poly(methyl methacrylate) or othermaterials may be used, including polycarbonate, cyclic olefin co-polymer(COC), glass, polyvinyl chloride, polyethylene, polyamide,polypropylene, polysulfone, polyurethane, and co-polymers of these andother polymers. Preferably at least the retentate member is fabricatedfrom a transparent material so that the cells can be visualized (see,e.g., FIG. 4Y and the description thereof). For example, a video cameramay be used to monitor cell growth by, e.g., density change measurementsbased on an image of an empty well, with phase contrast, or if, e.g., achromogenic marker, such as a chromogenic protein, is used to add adistinguishable color to the cells. Chromogenic markers such as blitzenblue, dreidel teal, virginia violet, vixen purple, prancer purple,tinsel purple, maccabee purple, donner magenta, cupid pink, seraphinapink, scrooge orange, and leor orange (the Chromogenic Protein Paintbox,all available from ATUM (Newark, Calif.)) obviate the need to usefluorescence, although fluorescent cell markers, fluorescent proteins,and chemiluminescent cell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growthfor, e.g., mammalian cells may be monitored by, e.g., the growth monitorsold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One,11(2):e0148469 (2016)). Further, automated colony pickers may beemployed, such as those sold by, e.g., TECAN (Pickolo™ system,Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.);Molecular Devices (QPix 400™ system, San Jose, Calif.); and SingerInstruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 450 may be controlled by, e.g., moving heated air over the top of(e.g., retentate member) of the SWIIN module 450, or by applying atransparent heated lid over at least the serpentine channel portion 460b of the retentate member 404. See, e.g., FIG. 4Y and the descriptionthereof infra.

As with the embodiments described previously, in SWIIN module 450 cellsand medium—at a dilution appropriate for Poisson or substantial Poissondistribution of the cells in the microwells of the perforated member—areflowed into serpentine channel 460 b from ports in retentate member 404,and the cells settle in the microwells while the medium passes throughthe filter into serpentine channel 460 a in permeate member 408. Thecells are retained in the microwells of perforated member 401 as thecells cannot travel through filter 403. Appropriate medium may beintroduced into permeate member 408 through permeate ports 411. Themedium flows upward through filter 403 to nourish the cells in themicrowells (perforations) of perforated member 401. Additionally, bufferexchange can be effected by cycling medium through the retentate andpermeate members. In operation, the cells are deposited into themicrowells, are grown for an initial, e.g., 2-100 doublings, editing isinduced by, e.g., raising the temperature of the SWIIN to 42° C. toinduce a temperature inducible promoter or by removing growth mediumfrom the permeate member and replacing the growth medium with a mediumcomprising a chemical component that induces an inducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module450 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 460 a and thus to filter 403 and pooled. Alternatively, ifcherry picking is desired, the growth of the cell colonies in themicrowells is monitored, and slow-growing colonies are directlyselected; or, fast-growing colonies are eliminated.

FIG. 4T is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 4T, it canbe seen that serpentine channel 460 a is disposed on the top of permeatemember 408 is defined by raised portions 476 and traverses permeatemember 408 for most of the length and width of permeate member 408except for the portion of permeate member 408 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 452can be seen). Moving from left to right, reservoir gasket 458 isdisposed upon the integrated reservoir cover 478 (cover not seen in thisFIG. 4T) of retentate member 404. Gasket 458 comprises reservoir accessapertures 432 a, 432 b, 432 c, and 432 d, as well as pneumatic ports 433a, 433 b, 433 c and 433 d. Also at the far left end is support 470.Disposed under permeate reservoir 452 can be seen one of two reservoirseals 462. In addition to the retentate member being in cross section,the perforated member 401 and filter 403 (filter 403 is not seen in thisFIG. 4T) are in cross section. Note that there are a number ofultrasonic tabs 464 disposed at the right end of SWIIN module 450 and onraised portion 476 which defines the channel turns of serpentine channel460 a, including ultrasonic tabs 464 extending through through-holes 466of perforated member 401. There is also a support 470 at the end distalreservoirs 452, 454 of permeate member 408.

FIG. 4U is a side perspective view of an assembled SWIIIN module 450,including, from right to left, reservoir gasket 458 disposed uponintegrated reservoir cover 478 (not seen) of retentate member 404.Gasket 458 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 458 comprises reservoir access apertures 432 a, 432 b, 432 c, and432 d, as well as pneumatic ports 433 a, 433 b, 433 c and 433 d. Also atthe far-left end is support 470 of permeate member 408. In addition,permeate reservoir 452 can be seen, as well as one reservoir seal 462.At the far-right end is a second support 470.

FIG. 4V is a side perspective view of the reservoir portion of permeatemember 408 and retentate member 404, including gasket 458. Seen arepermeate reservoirs 452 as the outside reservoirs, and retentatereservoirs 454 between permeate reservoirs 452. It should be apparent toone of ordinary skill in the art given the present description, however,that this particular configuration of reservoirs may be changed withpermeate 452 and retentate 454 reservoirs alternating in position; withboth permeate reservoirs 452 on one side of SWIIN module 450 and bothretentate reservoirs 454 on the other side of SWIIN module 450, or theretentate reservoirs 454 may be positioned at the two sides with thepermeate reservoirs 452 between the retentate reservoirs. Again, gasket458 comprises reservoir access apertures 432 a, 432 b, 432 c, and 432 d,as well as pneumatic ports 433 a, 433 b, 433 c and 433 d. In addition,two reservoir seals 462 can be seen, each sealing one permeate reservoir452 and one retentate reservoir 454. Also seen is support 470 at the“reservoir end” of permeate member 408.

FIG. 4W is a side perspective cross sectional view of permeate reservoir452 of permeate member 408 and retentate member 404 and gasket 458.Reservoir access aperture 432 c and pneumatic aperture 433 c can beseen, as well as support 470. Also seen is perforated member 401 andfilter 403 (filter 403 is not seen clearly in this FIG. 4W but issandwiched in between perforated member 401 and permeate member 408). Afluid path 472 from permeate reservoir 452 to serpentine channel 460 ain permeate member 408 can be seen, as can reservoir seal 462.

FIG. 4X is a small segment of a cross section of SWIIN module 450,showing the retentate member 404, perforated member 401, filter 403, andretentate member 408. FIG. 4X also shows a fluid path 472 from apermeate reservoir to the serpentine channel 460 a disposed in permeatemember 408, and a fluid path 474 from a retentate reservoir to theserpentine channel 460 b disposed in permeate member 404. As mentionedpreviously, the reservoir architecture of this embodiment isparticularly advantageous as bubbling is minimized. That is, because thereservoirs and reservoir ports are positioned below the retentate andpermeate serpentine channels, there is no instantaneous flow of fluid inthe reservoirs into channels that connect the reservoir ports to theretentate and permeate channels. Instead, flow is controlled by theapplication of pressure (positive or negative) and an appropriate timechosen by the user.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (top plate) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, or recovercells from specific wells (e.g., slow-growing cell colonies);alternatively, wells containing fast-growing cells can be identified andareas of UV light covering the fast-growing cell colonies can beprojected (or rastered with shutters) onto the SWIIN to irradiate orinhibit growth of those cells. Imaging may also be used to assure properfluid flow in the serpentine channel 460.

FIG. 4Y depicts the embodiment of the SWIIN module in FIGS. 4Q-4Xfurther comprising a heat management system including a heater and aheated cover. The heater cover facilitates the condensation managementthat is required for imaging. Assembly 498 comprises a SWIIN module 450seen lengthwise in cross section, where one permeate reservoir 452 isseen. Disposed immediately upon SWIIN module 450 is cover 494 anddisposed immediately below SWIIN module 450 is backlight 480, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 482, which is disposed over a heatsink 484. In thisFIG. 4Y, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 486 and heat sink 488, as well as twothermoelectric coolers 492, and a controller 490 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells (prokaryotic and eukaryotic) aswell as strains of cells that are, e.g., temperature sensitive, etc.,and allows use of temperature-sensitive promoters. Temperature controlallows for protocols to be adjusted to account for differences intransformation efficiency, cell growth and viability.

FIG. 4Z is an exemplary pneumatic block diagram suitable for the SWIINmodule depicted in FIGS. 4Q-4Y. In this configuration, there are twomanifold arms that are controlled independently, the pressure regulatorand vacuum regulator seen in FIG. 4P have been eliminated, and there aretwo proportional valves instead of one, one each for the manifold arms.Tables 3-5 relate to the pneumatic diagram in FIG. 4Z. Table 3 lists,for each step 1-32, the manifold arm status (open=arm open, closed=armclosed, motor engaged for pressurization); pump status (1: on, 0: off);energy status (1: energized, 0: de-energized) for each solenoid valve1-4; and the pressure in psi for each proportional valve. Table 4 lists,for each step 1-32, the detection and threshold status for flow meters 1and 2 as well as the duration of each step. When a change in pressureprecedes a valve event, there is a delay of 1 second after reaching theset point before energizing the valves to avoid applying over- andunder-shoots to the system. FALL=monitor for a falling signal,RISE=monitor for a rising signal. “Requires pLLD”=requirespressure-driven liquid level detection, such as, e.g., viaair-displacement pipettor. Table 5 lists, for each step 1-32, thevolumes for each reservoir, permeate reservoirs 1 and 2, and retentatereservoirs 1 and 2; the temperature of the SWIIN; and notes foroperation.

Automated Cell Editing Instruments and Modules

Automated Cell Editing Instruments

FIG. 5A depicts an exemplary stand-alone automated multi-module cellprocessing instrument 500 to, e.g., perform one of the exemplaryworkflows described infra, where the automated multi-module cellprocessing instrument performs the processes of cell growth, cellconcentration and buffer exchange to render the cells electrocompetent,cell transformation, cell selection, and cell editing all without humanintervention. The instrument 500, for example, may be and preferably isdesigned as a stand-alone desktop instrument for use within a laboratoryenvironment. The instrument 500 may incorporate a mixture of reusableand disposable components for performing the various integratedprocesses in conducting automated genome cleavage and/or editing incells. Illustrated is a gantry 502, providing an automated mechanicalmotion system (actuator) (not shown) that supplies XYZ axis motioncontrol to, e.g., an automated (i.e., robotic) liquid handling system558 including, e.g., an air displacement pipettor 532 which allows forcell processing among multiple modules without human intervention. Insome automated multi-module cell processing instruments, the airdisplacement pipettor 532 is moved by gantry 502 and the various modulesand reagent cartridges remain stationary; however, in other embodiments,the liquid handling system 558 may stay stationary while the variousmodules and reagent cartridges are moved. Also included in the automatedmulti-module cell processing instrument 500 is reagent cartridge 510comprising reservoirs 512 and transformation module 530 (e.g., aflow-through electroporation device as described in detail in relationto FIGS. 8A-8E), as well as a wash cartridge 504 comprising reservoirs506. The wash cartridge 504 may be configured to accommodate largetubes, for example, wash solutions, or solutions that are used oftenthroughout an iterative process. In one example, wash cartridge 504 maybe configured to remain in place when two or more reagent cartridges 510are sequentially used and replaced. Although reagent cartridge 510 andwash cartridge 504 are shown in FIG. 5A as separate cartridges, thecontents of wash cartridge 504 may be incorporated into reagentcartridge 510. The reagent cartridge 510 and wash cartridge 504 may beidentical except for the consumables (reagents or other componentscontained within the various inserts) inserted therein. Note in thisembodiment transformation module 530 is contained within reagentcartridge 510; however, in alternative embodiments transformation module530 is contained within its own module or may be part of another module,such as a growth module.

In some implementations, the wash and reagent cartridges 504 and 510comprise disposable kits (one or more of the various inserts andreagents) provided for use in the automated multi-module cellprocessing/editing instrument 500. For example, a user may open andposition each of the reagent cartridge 510 and the wash cartridge 504comprising various desired inserts and reagents within a chassis of theautomated multi-module cell editing instrument 500 prior to activatingcell processing.

Also illustrated in FIG. 5A is the robotic liquid handling system 558including the gantry 502 and air displacement pipettor 532. In someexamples, the robotic handling system 558 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips may be provided in a pipettetransfer tip supply (not shown) for use with the air displacementpipettor 532.

Inserts or components of the wash and reagent cartridges 504, 510, insome implementations, are marked with machine-readable indicia (notshown), such as bar codes, for recognition by the robotic handlingsystem 558. For example, the robotic liquid handling system 558 may scanone or more inserts within each of the wash and reagent cartridges 504,510 to confirm contents. In other implementations, machine-readableindicia may be marked upon each wash and reagent cartridge 504, 510, anda processing system (not shown, but see element 526 of FIG. 5B) of theautomated multi-module cell editing instrument 500 may identify a storedmaterials map based upon the machine-readable indicia. The exemplaryautomated multi-module cell processing instrument 500 of FIG. 5A furthercomprises a cell growth module 534. (Note, all modules recited brieflyhere are described in greater detail below.) In the embodimentillustrated in FIG. 5A, the cell growth module 534 comprises two cellgrowth vials 518, 520 (described in greater detail below in relation toFIGS. 6A-6D) as well as a cell concentration module 522 (described indetail in relation to FIGS. 7A-7K). In alternative embodiments, the cellconcentration module 522 may be separate from cell growth module 534,e.g., in a separate, dedicated module. Also illustrated as part of theautomated multi-module cell processing instrument 500 of FIG. 5A is anisolation module 540, served by, e.g., robotic liquid handing system 558and air displacement pipettor 532. Also seen are an optional nucleicacid assembly/desalting module 514 comprising a reaction chamber or tubereceptacle (not shown) and a magnet 516 to allow for purification ofnucleic acids using, e.g., magnetic solid phase reversibleimmobilization (SPRI) beads (Applied Biological Materials Inc.,Richmond, BC. The cell growth module, cell concentration module,transformation module, reagent cartridge, and nucleic acid assemblymodule are described in greater detail infra, and an exemplary isolationmodule (which may also serve as a recovery and growth module as well asan incubation and normalization module) is described in detail inrelation to FIGS. 3A-3J and 4A-4Y supra.

FIG. 5B is a plan view of the front of the exemplary multi-module cellprocessing instrument 500 depicted in FIG. 5A. Cartridge-based sourcematerials (such as in reagent cartridge 510), for example, may bepositioned in designated areas on a deck 502 of the instrument 500 foraccess by a robotic handling instrument (not shown in this figure). Asillustrated in FIG. 5B, the deck may include a protection sink 503 suchthat contaminants spilling, dripping, or overflowing from any of themodules of the instrument 500 are contained within a lip of theprotection sink 503. In addition to reagent cartridge 510, also seen inFIG. 5B is wash cartridge 504, isolation module 540, and a portion ofgrowth module 534. Also seen in this view is touch screen display 550,transformation module controls 538, electronics rack 536, and processingsystem 526.

FIGS. 5C through 5D illustrate side and front views, respectively, ofmulti-module cell processing instrument 500 comprising chassis 590 foruse in desktop versions of the automated multi-module cell editinginstrument 500. For example, the chassis 590 may have a width of about24-48 inches, a height of about 24-48 inches and a depth of about 24-48inches. Chassis 590 may be and preferably is designed to hold allmodules and disposable supplies used in automated cell processing and toperform all processes required without human intervention (that is,chassis 590 is configured to provide an integrated, stand-aloneautomated multi-module cell processing instrument). Chassis 590 maymount a robotic liquid handling system 558 for moving materials betweenmodules. As illustrated in FIG. 5C, the chassis 590 includes a cover 552having a handle 554 and hinge 556 a (hinges 556 b and 556 c are seen inFIG. 5D) for lifting the cover 552 and accessing the interior of thechassis 590. A cooling grate 564 (FIG. 5C) allows for air flow via aninternal fan (not shown). Further, the chassis 590 is lifted byadjustable feet 570 a, 570 c (feet 570, 570 b are shown in FIG. 5D).Adjustable feet 570 a-570 c, for example, may provide additional airflow beneath the chassis 590. A control button 566, in some embodiments,allows for single-button automated start and/or stop of cell processingwithin the automated multi-module cell processing instrument 500.

Inside the chassis 590, in some implementations, a robotic liquidhandling system 558 is disposed along a gantry 502 above wash cartridge504 (reagent cartridge 510 is not seen in these figures). Controlcircuitry, liquid handling tubes, air pump controls, valves, thermalunits (e.g., heating and cooling units) and other control mechanisms, insome embodiments, are disposed below a deck of the chassis 590, in acontrol box region 568. Also seen in both FIGS. 5C and 5D is isolationdevice or module 540. Nucleic acid assembly module 514 comprising amagnet 516 is seen in FIG. 5D.

Although not illustrated, in some embodiments a display screen may bepositioned on the front face of the chassis 590, for example covering aportion of the cover (e.g., see display 550 in FIG. 5B). The displayscreen may provide information to the user regarding the processingstatus of the automated multi-module cell editing instrument 500. Inanother example, the display screen may accept inputs from the user forconducting the cell processing.

The Rotating Growth Module

FIG. 6A shows one embodiment of a rotating growth vial 600 for use withthe cell growth device described herein. The rotating growth vial 600 isan optically-transparent container having an open end 604 for receivingliquid media and cells, a central vial region 606 that defines theprimary container for growing cells, a tapered-to-constricted region 618defining at least one light path 610, a closed end 616, and a driveengagement mechanism 612. The rotating growth vial 600 has a centrallongitudinal axis 620 around which the vial rotates, and the light path610 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 610 is positioned in the lower constricted portion ofthe tapered-to-constricted region 618. Optionally, some embodiments ofthe rotating growth vial 600 have a second light path 608 in the taperedregion of the tapered-to-constricted region 618. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 610 is shorter than the second light path 608 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 608 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 612 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 612 such that the rotating growth vial 600 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 600 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 600 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 600 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 600 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 600 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 604with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 604 mayoptionally include an extended lip 602 to overlap and engage with thecell growth device. In automated systems, the rotating growth vial 600may be tagged with a barcode or other identifying means that can be readby a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth vial 600 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 600 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 600 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 600. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 600 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 6B is a perspective view of one embodiment of a cell growth device630. FIG. 6C depicts a cut-away view of the cell growth device 630 fromFIG. 6B. In both figures, the rotating growth vial 600 is seenpositioned inside a main housing 636 with the extended lip 602 of therotating growth vial 600 extending above the main housing 636.Additionally, end housings 652, a lower housing 632 and flanges 634 areindicated in both figures. Flanges 634 are used to attach the cellgrowth device 630 to heating/cooling means or other structure (notshown). FIG. 6C depicts additional detail. In FIG. 6C, upper bearing 642and lower bearing 640 are shown positioned within main housing 636.Upper bearing 642 and lower bearing 640 support the vertical load ofrotating growth vial 600. Lower housing 632 contains the drive motor638. The cell growth device 630 of FIG. 6C comprises two light paths: aprimary light path 644, and a secondary light path 650. Light path 644corresponds to light path 610 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 600, andlight path 650 corresponds to light path 608 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth via 6001.Light paths 610 and 608 are not shown in FIG. 6C but may be seen in FIG.6A. In addition to light paths 644 and 640, there is an emission board648 to illuminate the light path(s), and detector board 646 to detectthe light after the light travels through the cell culture liquid in therotating growth vial 600.

The motor 638 engages with drive mechanism 612 and is used to rotate therotating growth vial 600. In some embodiments, motor 638 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 638 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 636, end housings 652 and lower housing 632 of the cellgrowth device 630 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 600 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 630 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 630 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 630—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 630, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 6D illustrates a cell growth device 630 as part of an assemblycomprising the cell growth device 630 of FIG. 6B coupled to light source690, detector 692, and thermal components 694. The rotating growth vial600 is inserted into the cell growth device. Components of the lightsource 690 and detector 692 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 632 that houses the motor that rotatesthe rotating growth vial 600 is illustrated, as is one of the flanges634 that secures the cell growth device 630 to the assembly. Also, thethermal components 694 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 630 to the thermal components 694 via the flange 634 on the baseof the lower housing 632. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 600 is controlled to approximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 600 by piercing though the foil seal orfilm. The programmed software of the cell growth device 630 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 600. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 600 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring systemtakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 630 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 630 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 630 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.

Cell Concentration Module

FIGS. 7A-7K depict variations on one embodiment of a cellconcentration/buffer exchange cassette and module that utilizestangential flow filtration. One embodiment of a cell concentrationdevice described herein operates using tangential flow filtration (TFF),also known as crossflow filtration, in which the majority of the feedflows tangentially over the surface of the filter thereby reducing cake(retentate) formation as compared to dead-end filtration, in which thefeed flows into the filter. Secondary flows relative to the main feedare also exploited to generate shear forces that prevent filter cakeformation and membrane fouling thus maximizing particle recovery, asdescribed below.

The TFF device described herein was designed to take into account twoprimary design considerations. First, the geometry of the TFF deviceleads to filtering the cell culture over a large surface area so as tominimize processing time. Second, the design of the TFF device isconfigured to minimize filter fouling. FIG. 7A is a general model 750 oftangential flow filtration. The TFF device operates using tangentialflow filtration, also known as cross-flow filtration. FIG. 7A showscells flowing over a membrane 754, where the feed flow of the cells 752in medium or buffer is parallel to the membrane 754. TFF is differentfrom dead-end filtration where both the feed flow and the pressure dropare perpendicular to a membrane or filter.

FIG. 7B depicts a top view of the lower member of one embodiment of aTFF device/module providing tangential flow filtration. As can be seenin the embodiment of the TFF device of FIG. 7B, TFF device 700 comprisesa channel structure 716 comprising a flow channel 702 b through which acell culture is flowed. The channel structure 716 comprises a singleflow channel 702 b that is horizontally bifurcated by a membrane (notshown) through which buffer or medium may flow, but cells cannot. Thisparticular embodiment comprises an undulating serpentine geometry 714(i.e., the small “wiggles” in the flow channel 702) and a serpentine“zig-zag” pattern where the flow channel 702 crisscrosses the devicefrom one end at the left of the device to the other end at the right ofthe device. The serpentine pattern allows for filtration over a highsurface area relative to the device size and total channel volume, whilethe undulating contribution creates a secondary inertial flow to enableeffective membrane regeneration preventing membrane fouling. Although anundulating geometry and serpentine pattern are exemplified here, otherchannel configurations may be used as long as the channel can bebifurcated by a membrane, and as long as the channel configurationprovides for flow through the TFF module in alternating directions. Inaddition to the flow channel 702 b, portals 704 and 706 as part of thechannel structure 716 can be seen, as well as recesses 708. Portals 704collect cells passing through the channel on one side of a membrane (notshown) (the “retentate”), and portals 706 collect the medium (“filtrate”or “permeate”) passing through the channel on the opposite side of themembrane (not shown). In this embodiment, recesses 708 accommodatescrews or other fasteners (not shown) that allow the components of theTFF device to be secured to one another.

The length 710 and width 712 of the channel structure 716 may varydepending on the volume of the cell culture to be grown and the opticaldensity of the cell culture to be concentrated. The length 710 of thechannel structure 716 typically is from 1 mm to 300 mm, or from 50 mm to250 mm, or from 60 mm to 200 mm, or from 70 mm to 150 mm, or from 80 mmto 100 mm. The width of the channel structure 716 typically is from 1 mmto 120 mm, or from 20 mm to 100 mm, or from 30 mm to 80 mm, or from 40mm to 70 mm, or from 50 mm to 60 mm. The cross-section configuration ofthe flow channel 702 may be round, elliptical, oval, square,rectangular, trapezoidal, or irregular. If square, rectangular, oranother shape with generally straight sides, the cross section may befrom about 10 μm to 1000 μm wide, or from 200 μm to 800 μm wide, or from300 μm to 700 μm wide, or from 400 μm to 600 μm wide; and from about 10μm to 1000 μm high, or from 200 μm to 800 μm high, or from 300 μm to 700μm high, or from 400 μm to 600 μm high. If the cross section of the flowchannel 602 is generally round, oval or elliptical, the radius of thechannel may be from about 50 μm to 1000 μm in hydraulic radius, or from5 μm to 800 μm in hydraulic radius, or from 200 μm to 700 μm inhydraulic radius, or from 300 μm to 600 μm wide in hydraulic radius, orfrom about 200 to 500 μm in hydraulic radius.

When looking at the top view of the TFF device/module of FIG. 7B, notethat there are two retentate portals 704 and two filtrate portals 706,where there is one of each type portal at both ends (e.g., the narrowedge) of the device 700. In other embodiments, retentate and filtrateportals can on the same surface of the same member (e.g., upper or lowermember), or they can be arranged on the side surfaces of the assembly.Unlike other TFF devices that operate continuously, the TFFdevice/module described herein uses an alternating method forconcentrating cells. The overall work flow for cell concentration usingthe TFF device/module involves flowing a cell culture or cell sampletangentially through the channel structure. The membrane bifurcating theflow channels retains the cells on one side of the membrane and allowsunwanted medium or buffer to flow across the membrane into a filtrateside (e.g., lower member 720) of the device. In this process, a fixedvolume of cells in medium or buffer is driven through the device untilthe cell sample is collected into one of the retentate portals 704, andthe medium/buffer that has passed through the membrane is collectedthrough one or both of the filtrate portals 706. All types ofprokaryotic and eukaryotic cells—both adherent and non-adherentcells—can be grown in the TFF device. Adherent cells may be grown onbeads or other cell scaffolds suspended in medium that flow through theTFF device.

In the cell concentration process, passing the cell sample through theTFF device and collecting the cells in one of the retentate portals 704while collecting the medium in one of the filtrate portals 706 isconsidered “one pass” of the cell sample. The transfer between retentatereservoirs “flips” the culture, The retentate and filtrate portalscollecting the cells and medium, respectively, for a given pass resideon the same end of TFF device/module 700 with fluidic connectionsarranged so that there are two distinct flow layers for the retentateand filtrate sides, but if the retentate portal 704 resides on the uppermember of device/module 700 (that is, the cells are driven through thechannel above the membrane and the filtrate (medium) passes to theportion of the channel below the membrane), the filtrate portal 706 willreside on the lower member of device/module 100 and vice versa (that is,if the cell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). This configuration can be seen more clearly in FIGS. 7C-7D,where the retentate flows 760 from the retentate portals 704 and thefiltrate flows 770 from the filtrate portals 706.

At the conclusion of a “pass” in the growth concentration process, thecell sample is collected by passing through the retentate portal 704 andinto the retentate reservoir (not shown). To initiate another “pass”,the cell sample is passed again through the TFF device, this time in aflow direction that is reversed from the first pass. The cell sample iscollected by passing through the retentate portal 704 and into retentatereservoir (not shown) on the opposite end of the device/module from theretentate portal 704 that was used to collect cells during the firstpass. Likewise, the medium/buffer that passes through the membrane onthe second pass is collected through the filtrate portal 706 on theopposite end of the device/module from the filtrate portal 706 that wasused to collect the filtrate during the first pass, or through bothportals. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been concentrated to a desired volume, and both filtrateportals can be open during the passes to reduce operating time. Inaddition, buffer exchange may be effected by adding a desired buffer (orfresh medium) to the cell sample in the retentate reservoir, beforeinitiating another “pass”, and repeating this process until the oldmedium or buffer is diluted and filtered out and the cells reside infresh medium or buffer. Note that buffer exchange and cell concentrationmay (and typically do) take place simultaneously.

FIG. 7C depicts a top view of upper (722) and lower (720) members of anexemplary TFF module. Again, portals 704 and 706 are seen. As notedabove, recesses—such as the recesses 708 seen in FIG. 7B—provide a meansto secure the components (upper member 722, lower member 720, andmembrane 724) of the TFF device/membrane to one another during operationvia, e.g., screws or other like fasteners. However, in alterativeembodiments an adhesive, such as a pressure sensitive adhesive, orultrasonic welding, or solvent bonding, may be used to couple the uppermember 722, lower member 720, and membrane 724 together. Indeed, one ofordinary skill in the art given the guidance of the present disclosurecan find yet other configurations for coupling the components of the TFFdevice, such as e.g., clamps; mated fittings disposed on the upper andlower members; combination of adhesives, welding, solvent bonding, andmated fittings; and other such fasteners and couplings.

Note that there is one retentate portal and one filtrate portal on each“end” (e.g., the narrow edges) of the TFF device/module. The retentateand filtrate portals on the left side of the device/module will collectcells (flow path at 760) and medium (flow path at 770), respectively,for the same pass. Likewise, the retentate and filtrate portals on theright side of the device/module will collect cells (flow path at 760)and medium (flow path at 770), respectively, for the same pass. In thisembodiment, the retentate is collected from portals 704 on the topsurface of the TFF device, and filtrate is collected from portals 706 onthe bottom surface of the device. The cells are maintained in the TFFflow channel above the membrane 724, while the filtrate (medium) flowsthrough membrane 724 and then through portals 706; thus, thetop/retentate portals and bottom/filtrate portals configuration ispractical. It should be recognized, however, that other configurationsof retentate and filtrate portals may be implemented such as positioningboth the retentate and filtrate portals on the side (as opposed to thetop and bottom surfaces) of the TFF device. In FIG. 7C, the channelstructure 702 b can be seen on the bottom member 720 of the TFF device700. However, in other embodiments, retentate and filtrate portals canreside on the same of the TFF device.

Also seen in FIG. 7C is membrane or filter 724. Filters or membranesappropriate for use in the TFF device/module are those that are solventresistant, are contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.2 μm, however for other cell types, the pore sizes can be ashigh as 5 μm. Indeed, the pore sizes useful in the TFF device/moduleinclude filters with sizes from 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48μm, 0.49 μm, 0.50 μm and larger. The filters may be fabricated from anysuitable non-reactive material including cellulose mixed ester(cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, glass fiber, or metal substratesas in the case of laser or electrochemical etching. The TFF device shownin FIGS. 7C and 7D do not show a seat in the upper 712 and lower 720members where the filter 724 can be seated or secured (for example, aseat half the thickness of the filter in each of upper 712 and lower 720members); however, such a seat is contemplated in some embodiments.

FIG. 7D depicts a bottom view of upper and lower components of theexemplary TFF module shown in FIG. 7C. FIG. 7D depicts a bottom view ofupper (722) and lower (720) components of an exemplary TFF module. Againportals 704 and 706 are seen. Note again that there is one retentateportal and one filtrate portal on each end of the device/module. Theretentate and filtrate portals on the left side of the device/modulewill collect cells (flow path at 760) and medium (flow path at 770),respectively, for the same pass. Likewise, the retentate and filtrateportals on the right side of the device/module will collect cells (flowpath at 760) and medium (flow path at 770), respectively, for the samepass. In FIG. 7D, the channel structure 702 a can be seen on the uppermember 722 of the TFF device 700. Thus, looking at FIGS. 7C and 7D, notethat there is a channel structure 702 (702 a and 702 b) in both theupper and lower members, with a membrane 724 between the upper and lowerportions of the channel structure. The channel structure 702 of theupper 722 and lower 720 members (702 a and 702 b, respectively) mate tocreate the flow channel with the membrane 624 positioned horizontallybetween the upper and lower members of the flow channel therebybifurcating the flow channel.

Medium exchange (during cell growth) or buffer exchange (during cellconcentration or rendering the cells competent) is performed on the TFFdevice/module by adding fresh medium to growing cells or a desiredbuffer to the cells concentrated to a desired volume; for example, afterthe cells have been concentrated at least 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold,200-fold or more. A desired exchange medium or exchange buffer is addedto the cells either by addition to the retentate reservoir or thoroughthe membrane from the filtrate side and the process of passing the cellsthrough the TFF device 700 is repeated until the cells have been grownto a desired optical density or concentrated to a desired volume in theexchange medium or buffer. This process can be repeated any number ofdesired times so as to achieve a desired level of exchange of the bufferand a desired volume of cells. The exchange buffer may comprise, e.g.,glycerol or sorbitol thereby rendering the cells competent fortransformation in addition to decreasing the overall volume of the cellsample.

The TFF device 700 may be fabricated from any robust material in whichchannels (and channel branches) may be milled including stainless steel,silicon, glass, aluminum, or plastics including cyclic-olefin copolymer(COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module is thermally-conductive so that thecell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto this mass production techniques.

FIGS. 7E-7K depict an alternative embodiment of a tangential flowfiltration (TFF) device/module, where the module has the advantage of areduced footprint, in, e.g., an automated multi-module cell processinginstrument. FIG. 7E depicts a configuration of an upper (retentate)member 7022 (on left), a membrane or filter 7024 (middle), and a lower(permeate/filtrate) member 7020 (on the right). In the configurationshown in FIGS. 7E-7K, the retentate member 7022 is no longer “upper” andthe permeate/filtrate member 7020 is no longer “lower”, as the retentatemember 7022 and permeate/filtrate member 7020 are coupled side-to-sideas seen in FIGS. 7J and 7K. In FIG. 7E, retentate member 7022 comprisesa tangential flow channel 7002, which has a serpentine configurationthat initiates at one lower corner of retentate member 7022—specificallyat retentate port 7028—traverses across and up then down and acrossretentate member 7022, ending in the other lower corner of retentatemember 7022 at a second retentate port 7028. Also seen on retentatemember 7022 is energy director 7091, which circumscribes the regionwhere membrane or filter 7024 is seated. Energy director 7091 in thisembodiment mates with and serves to facilitate ultrasonic wending orbonding of retentate member 7022 with permeate/filtrate member 7020 viathe energy director component on permeate/filtrate member 7020. Alsoseen is membrane or filter 7024 has through-holes for retentate ports7028, which is configured to seat within the circumference of energydirectors 7091 between the retentate member 7022 and thepermeate/filtrate member 7020. Permeate/filtrate member 7020 comprises,in addition to energy director 7091, through-holes for retentate port7028 at each bottom corner (which mate with the through-holes forretentate ports 7028 at the bottom corners of membrane 7024 andretentate ports 7028 in retentate member 7022), as well as a tangentialflow channel 7002 and a single permeate/filtrate port 7026 positioned atthe top and center of permeate/filtrate member 7020. The tangential flowchannel 7002 structure in this embodiment has a serpentine configurationand an undulating geometry, although other geometries may be used. Insome aspects, the length of the tangential flow channel is from 10 mm to1000 mm, from 60 mm to 200 mm, or from 80 mm to 100 mm. In some aspects,the width of the channel structure is from 10 mm to 120 mm, from 40 mmto 70 mm, or from 50 mm to 60 mm. In some aspects, the cross section ofthe tangential flow channel 1202 is rectangular. In some aspects, thecross section of the tangential flow channel 1202 is 5 μm to 1000 μmwide and 5 μm to 1000 μm high, 300 μm to 700 μm wide and 300 μm to 700μm high, or 400 μm to 600 μm wide and 400 μm to 600 μm high. In otheraspects, the cross section of the tangential flow channel 1202 iscircular, elliptical, trapezoidal, or oblong, and is 100 μm to 1000 μmin hydraulic radius, 300 μm to 700 μm in hydraulic radius, or 400 μm to600 μm in hydraulic radius.

FIG. 7F is a side perspective view of a reservoir assembly 7050. Theembodiment of FIG. 7F, the retentate member is separate from thereservoir assembly. Reservoir assembly 7050 comprises retentatereservoirs 7052 on either side of a single permeate reservoir 7054.Retentate reservoirs 7052 are used to contain the cells and medium asthe cells are transferred through the cell concentration/growth deviceor module and into the retentate reservoirs during cell concentrationand/or growth. Permeate/filtrate reservoir 7054 is used to collect thefiltrate fluids removed from the cell culture during cell concentration,or old buffer or medium during cell growth. In the embodiment depictedin FIGS. 7E-7L, buffer or medium is supplied to the permeate/filtratemember from a reagent reservoir separate from the device module.Additionally seen in FIG. 7F are grooves 7032 to accommodate pneumaticports (not seen), permeate/filtrate port 7026, and retentate portthrough-holes 7028. The retentate reservoirs are fluidically coupled tothe retentate ports 7028, which in turn are fluidically coupled to theportion of the tangential flow channel disposed in the retentate member(not shown). The permeate/filtrate reservoir is fluidically coupled tothe permeate/filtrate port 7026 which in turn are fluidically coupled tothe portion of the tangential flow channel disposed in permeate/filtratemember (not shown), where the portions of the tangential flow channelsare bifurcated by membrane (not shown). In embodiments including thepresent embodiment, up to 120 mL of cell culture can be grown and/orfiltered, or up to 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30mL or 20 mL of cell culture can be grown and/or concentrated.

FIG. 7G depicts a top-down view of the reservoir assembly 7050 shown inFIG. 7F, FIG. 7H depicts a cover 7044 for reservoir assembly 7050 shownin FIGS. 7F, and 7I depicts a gasket 7045 that in operation is disposedon cover 7044 of reservoir assembly 7050 shown in FIG. 7F. FIG. 7G is atop-down view of reservoir assembly 7050, showing two retentatereservoirs 7052, one on either side of permeate reservoir 7054. Alsoseen are grooves 7032 that will mate with a pneumatic port (not shown),and fluid channels 7034 that reside at the bottom of retentatereservoirs 7052, which fluidically couple the retentate reservoirs 7052with the retentate ports 7028 (not shown), via the through-holes for theretentate ports in permeate/filtrate member 7220 and membrane 7024 (alsonot shown). FIG. 7H depicts a cover 7044 that is configured to bedisposed upon the top of reservoir assembly 7050. Cover 7044 has roundcut-outs at the top of retentate reservoirs 7052 and permeate/filtratereservoir 7054. Again, at the bottom of retentate reservoirs 7052 fluidchannels 7034 can be seen, where fluid channels 7034 fluidically coupleretentate reservoirs 7052 with the retentate ports 7028 (not shown).Also shown are three pneumatic ports 7030 for each retentate reservoir7052 and permeate/filtrate reservoir 7054. FIG. 7I depicts a gasket 7045that is configures to be disposed upon the cover 7044 of reservoirassembly 7050. Seen are three fluid transfer ports 7042 for eachretentate reservoir 7052 and for permeate/filtrate reservoir 7054.Again, three pneumatic ports 7030, for each retentate reservoir 7052 andfor permeate/filtrate reservoir 7054, are shown.

FIG. 7J depicts an exploded view of a TFF module 7000. Seen arecomponents reservoir assembly 7050, a cover 7044 to be disposed onreservoir assembly 7050, a gasket 7045 to be disposed on cover 7044,retentate member 7022, membrane or filter 7024, and permeate/filtratemember 7020. Also seen is permeate/filtrate port 7026, which mates withpermeate/filtrate port 7026 on permeate/filtrate reservoir 7054, as wellas two retentate ports 7028, which mate with retentate ports 7028 onretentate reservoirs 7052 (where only one retentate reservoir 7052 canbe seen clearly in this FIG. 7J). Also seen are through-holes forretentate ports 7028 in membrane 7024 and permeate/filtrate member 7020.

FIG. 7K depicts an embodiment of assembled TFF module 7000. Note that inthis embodiment of a TFF module the retentate member 7022 is no longer“upper”, and the permeate/filtrate member 7020 is no longer “lower”, asthe retentate member 7022 and permeate/filtrate member 7020 are coupledside-to-side with membrane 7024 sandwiched between retentate member 7022and permeate/filtrate member 7020. Also, retentate member 7022, membranemember 7024, and permeate/filtrate member 7020 are coupled side-to-sidewith reservoir assembly 7050. Seen are two retentate ports 7028 (whichcouple the tangential flow channel 7002 in retentate member 7022 to thetwo retentate reservoirs (not shown), and one permeate/filtrate port7026, which couples the tangential flow channel 7002 inpermeate/filtrate member 7020 to the permeate/filtrate reservoir (notshown). Also seen is tangential flow channel 7002, which is formed bythe mating of retentate member 7022 and permeate/filtrate member 7020,with membrane 7024 sandwiched between and bifurcating tangential flowchannel 7002. Also seen is energy director 7091, which in this FIG. 7Khas been used to ultrasonically weld or couple retentate member 7022 andpermeate/filtrate member 7020, surrounding membrane 7024. Cover 7044 canbe seen on top of reservoir assembly 7050, and gasket 7045 is disposedupon cover 7044. Gasket 7045 engages with and provides a fluid-tightseal and pneumatic connections with fluid transfer ports 7042 andpneumatic ports 7030, respectively. FIG. 7J also shows the length,height, and width dimensions of the TFF module 7000. The assembled TFFdevice 7000 typically is from 50 to 175 mm in height, or from 75 to 150mm in height, or from 90 to 120 mm in height; from 50 to 175 mm inlength, or from 75 to 150 mm in length, or from 90 to 120 mm in length;and is from 30 to 90 mm in depth, or from 40 to 75 mm in depth, or fromabout 50 to 60 mm in depth. An exemplary TFF device is 110 mm in height,120 mm in length, and 55 mm in depth.

Like in other embodiments described herein, the TFF device or moduledepicted in FIGS. 7E-7K can constantly measure cell culture growth, andin some aspects cell culture growth is measured via optical density (OD)of the cell culture in one or both of the retentate reservoirs and/or inthe flow channel of the TFF device. Optical density may be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or so on minutes. Further, the TFF module can adjust growthparameters (temperature, aeration) to have the cells at a desiredoptical density at a desired time.

FIG. 7L is an exemplary pneumatic block diagram suitable for the TFFmodule depicted in FIGS. 7E-7K. The pump is connected to two solenoidvalves (SV5 and SV6) delivering positive pressure (P) or negativepressure (V). The two solenoid valves SV5 and SV6 couple the pump to themanifold, and two solenoid valves, SV1 and SV2, are connected to thereservoirs (RR1 and RR2). There are also two solenoid valves in reserve(SV3 and SV4). There is a proportional valve (PV2 and PV2), a flow meter(FM1 and FM2), and a pressure sensor (Pressure Sensors 1 and 2)positioned in between each of solenoid valves SV1 and SV2 connecting thepump to the system and the solenoid valves SV1 And SV2 to thereservoirs. The pressure sensors and prop valves work in concert in afeedback loop to maintain the required pressure.

As an alternative to the TFF module described above, a cellconcentration module comprising a hollow filter may be employed.Examples of filters suitable for use in the present invention includemembrane filters, ceramic filters and metal filters. The filter may beused in any shape; the filter may for example be cylindrical oressentially flat. Preferably, the filter used is a membrane filter,preferably a hollow fiber filter. The term “hollow fiber” is meant atubular membrane. The internal diameter of the tube is at least 0.1 mm,more preferably at least 0.5 mm, most preferably at least 0.75 mm andpreferably the internal diameter of the tube is at most 10 mm, morepreferably at most 6 mm, most preferably at most 1 mm. Filter modulescomprising hollow fibers are commercially available from variouscompanies, including G.E. Life Sciences (Marlborough, Mass.) andInnovaPrep (Drexel, Mo.). Specific examples of hollow fiber filtersystems that can be used, modified or adapted for use in the presentmethods and systems include, but are not limited to, U.S. Pat. Nos.9,738,918; 9,593,359; 9,574,977; 9,534,989; 9,446,354; 9,295,824;8,956,880; 8,758,623; 8,726,744; 8,677,839; 8,677,840; 8,584,536;8,584,535; and 8,110,112.

Nucleic Acid Assembly Module

Certain embodiments of the automated multi-module cell editinginstruments of the present disclosure optionally include a nucleic acidassembly module. The nucleic acid assembly module is configured toaccept and assemble the nucleic acids necessary to facilitate thedesired genome editing events. In general, the term “vector” refers to anucleic acid molecule capable of transporting a desired nucleic acid towhich it has been linked into a cell. Vectors include, but are notlimited to, nucleic acid molecules that are single-stranded,double-stranded, or partially double-stranded; nucleic acid moleculesthat include one or more free ends, no free ends (e.g., circular);nucleic acid molecules that include DNA, RNA, or both; and othervarieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,where virally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors” or “editingvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. Additional vectors includefosmids, phagemids, and synthetic chromosomes.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transcription, and for some nucleic acid sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements—which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in US Pub. No. 2004/0171156, the contents of whichare herein incorporated by reference in their entirety for all purposes.

In some embodiments, a regulatory element is operably linked to one ormore elements of a targetable nuclease system so as to drivetranscription, and for some nucleic acid sequences, translation andexpression of the one or more components of the targetable nucleasesystem.

In addition, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can beyeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably likedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated multi-modulecell editing instruments include, but are not limited to, those assemblymethods that use restriction endonucleases, including PCR, BioBrickassembly (U.S. Pat. No. 9,361,427), Type IIS cloning (e.g., GoldenGateassembly, European Patent Application Publication EP 2 395 087 A1), andLigase Cycling Reaction (de Kok, ACS Synth Biol., 3(2):97-106 (2014);Engler, et al., PLoS One, 3(11):e3647 (2008); and U.S. Pat. No.6,143,527). In other embodiments, the nucleic acid assembly techniquesperformed by the disclosed automated multi-module cell editinginstruments are based on overlaps between adjacent parts of the nucleicacids, such as isothermal nucleic acid assembly, CPEC, SLIC, LigaseCycling etc. Additional assembly methods include gap repair in yeast(Bessa, Yeast, 29(10):419-23 (2012)), gateway cloning (Ohtsuka, CurrPharm Biotechnol, 10(2):244-51 (2009)); U.S. Pat. Nos. 5,888,732; and6,277,608), and topoisomerase-mediated cloning (Udo, PLoS One,10(9):e0139349 (2015); and U.S. Pat. No. 6,916,632). These and othernucleic acid assembly techniques are described, e.g., in Sands andBrent, Curr Protoc Mol Biol., 113:3.26.1-3.26.20 (2016).

The nucleic acid assembly module is temperature controlled dependingupon the type of nucleic acid assembly used in the automatedmulti-module cell editing instrument. For example, when PCR is utilizedin the nucleic acid assembly module, the module includes a thermocyclingcapability allowing the temperatures to cycle between denaturation,annealing and extension steps. When single temperature assembly methods(e.g., isothermal assembly methods) are utilized in the nucleic acidassembly module, the module provides the ability to reach and hold atthe temperature that optimizes the specific assembly process beingperformed. These temperatures and the duration for maintaining thesetemperatures can be determined by a preprogrammed set of parametersexecuted by a script, or manually controlled by the user using theprocessing system of the automated multi-module cell editing instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction. Certain isothermalassembly methods can combine simultaneously up to 15 nucleic acidfragments based on sequence identity. The assembly method provides, insome embodiments, nucleic acids to be assembled which include anapproximate 20-40 base overlap with adjacent nucleic acid fragments. Thefragments are mixed with a cocktail of three enzymes—an exonuclease, apolymerase, and a ligase—along with buffer components. Because theprocess is isothermal and can be performed in a 1-step or 2-step methodusing a single reaction vessel, isothermal assembly reactions are idealfor use in an automated multi-module cell editing instrument. The 1-stepmethod allows for the assembly of up to five different fragments using asingle step isothermal process. The fragments and the master mix ofenzymes are combined and incubated at 50° C. for up to one hour. For thecreation of more complex constructs with up to fifteen fragments or forincorporating fragments from 100 bp up to 10 kb, typically the 2-step isused, where the 2-step reaction requires two separate additions ofmaster mix; one for the exonuclease and annealing step and a second forthe polymerase and ligation steps.

Transformation Module

FIGS. 8A-8E depict variations on one embodiment of a cell transformationmodule (in this case, a flow-through electroporation device) that may beincluded in a cell growth/concentration/transformation instrument. FIGS.8A and 8B are top perspective and bottom perspective views,respectively, of six co-joined flow-through electroporation devices 850.FIG. 8A depicts six flow-through electroporation units 850 arranged on asingle substrate 856. Each of the six flow-through electroporation units850 have inlet wells 852 that define cell sample inlets and outlet wells854 that define cell sample outlets. FIG. 8B is a bottom perspectiveview of the six co-joined flow-through electroporation devices of FIG.8A also depicting six flow-through electroporation units 850 arranged ona single substrate 856. Six inlet wells 852 can be seen, one for eachflow-through electroporation unit 850, and one outlet well 854 can beseen (the outlet well of the left-most flow-through electroporation unit850). Additionally seen in FIG. 8B are an inlet 802, outlet 804, flowchannel 806 and two electrodes 808 on either side of a constriction inflow channel 806 in each flow-through electroporation unit 850. Once thesix flow-through electroporation units 850 are fabricated, they can beseparated from one another (e.g., “snapped apart”) and used one at atime, or alternatively in embodiments where two or more flow-throughelectroporation units 850 can be used in parallel without separation.

The flow-through electroporation devices 850 achieve high efficiencycell electroporation with low toxicity. The flow-through electroporationdevices 850 of the disclosure allow for particularly easy integrationwith robotic liquid handling instrumentation that is typically used inautomated systems such as air displacement pipettors. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 10 mL and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit electroporation deviceconfigurations; and integrated, automatic, multi-module cell processingand analysis.

In specific embodiments of the flow-through electroporation devices 850of the disclosure, the toxicity level of the transformation results ingreater than 10% viable cells after electroporation, preferably greaterthan 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%,85%, 90%, or even 95% viable cells following transformation, dependingon the cell type and the nucleic acids being introduced into the cells.

The flow-through electroporation device 850 described in relation toFIGS. 8A-8E comprises a housing with an electroporation chamber, a firstelectrode and a second electrode configured to engage with an electricpulse generator, by which electrical contacts engage with the electrodesof the electroporation device 850. In certain embodiments, theelectroporation devices are autoclavable and/or disposable, and may bepackaged with reagents in a reagent cartridge. The electroporationdevice 850 may be configured to electroporate cell sample volumesbetween 1 μl to 2 mL, 10 μl to 1 mL, 25 μl to 750 μl, or 50 μl to 500μl. The cells that may be electroporated with the disclosedelectroporation devices 850 include mammalian cells (including humancells), plant cells, yeasts, other eukaryotic cells, bacteria, archaea,and other cell types.

In one exemplary embodiment, FIG. 8C depicts a top view of aflow-through electroporation device 850 having an inlet 802 forintroduction of cells and an exogenous reagent to be electroporated intothe cells (“cell sample”) and an outlet 804 for the cell samplefollowing electroporation. Electrodes 808 are introduced throughelectrode channels (not shown in this FIG. 8C) in the device. FIG. 8Dshows a cutaway view from the top of flow-through electroporation device850, with the inlet 802, outlet 804, and electrodes 808 positioned withrespect to a constriction in flow channel 806. A side cutaway view of alower portion of flow-through electroporation device 850 in FIG. 8Eillustrates that electrodes 808 in this embodiment are positioned inelectrode channels 810 and perpendicular to flow channel 806 such thatthe cell sample flows from the inlet channel 812 through the flowchannel 806 to the outlet channel 814, and in the process the cellsample flows into the electrode channels 810 to be in contact withelectrodes 808. In this aspect, the inlet channel 812, outlet channel814 and electrode channels 810 all originate from the top planar side ofthe device; however, the flow-through electroporation architecturedepicted in FIGS. 8C-8E is but one architecture useful with the reagentcartridges described herein. Additional electrode architectures aredescribed, e.g., in U.S. Ser. No. 16/147,120, filed 24 Sep. 2018; Ser.No. 16/147,865, filed 30 Sep. 2018; and Ser. No. 16/147,871, filed 30Sep. 2018.

Exemplary Workflows

FIG. 9 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising anisolation or substantial isolation/incubation/editing and normalizationor cherry-picking module for enrichment or selection of edited cells.The cell processing instrument 900 may include a housing 944, areservoir of cells to be transformed or transfected 902, and a growthmodule (a cell growth device) 904. The cells to be transformed aretransferred from a reservoir to the growth module to be cultured untilthe cells hit a target OD. Once the cells hit the target OD, the growthmodule may cool or freeze the cells for later processing, or the cellsmay be transferred to a cell concentration module 930 where the cellsare rendered electrocompetent and concentrated to a volume optimal forcell transformation. Exemplary cell concentration devices of use in theautomated multi-module cell processing system include those described inU.S. Ser. No. 62/728,365, filed 7 Sep. 2018; 62/857,599, filed 5 Jun.2019; and 62/867,415, filed 27 Jun. 2019, all of which are incorporatedby reference in their entirety. Once concentrated, the cells are thentransferred to the electroporation device 908 (e.g.,transformation/transfection module, with one exemplary module describedabove in relation to FIGS. 8A-8E). Exemplary electroporation devices ofuse in the automated multi-module cell processing instruments includeflow-through electroporation devices such as those described in U.S.Ser. No. 16/147,120, filed 28 Sep. 2018; Ser. No. 16/147,353, filed 28Sep. 2018; Ser. No. 16/147,865, filed 30 Sep. 2018; and Ser. No.16/147,871, filed 30 Sep. 2018 all of which are herein incorporated byreference in their entirety.

In addition to the reservoir for storing the cells 930, the automatedmulti-module cell processing instrument 900 may include a reservoir forstoring editing oligonucleotide cassettes 916 and a reservoir forstoring an expression vector backbone 918. Both the editingoligonucleotide cassettes and the expression vector backbone aretransferred from a reagent cartridge to a nucleic acid assembly module920, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 922 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 916 or 918. Once the processes carried out by thepurification module 922 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 908, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via cell concentration module 930. In electroporationdevice 908, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/selection module 910. For examples of multi-module cell editinginstruments, see U.S. Pat. No. 10,253,316, filed 30 Jun. 2018; U.S. Pat.No. 10,329,559, filed 7 Feb. 2019; and U.S. Pat. No. 10,323,242, filed 7Feb. 2019; and U.S. Ser. No. 16/412,175, filed 14 May 2019; Ser. No.16/412,195, filed 14 May 2019; and Ser. No. 16/423,289, filed 28 May2019, all of which are herein incorporated by reference in theirentirety.

Following recovery, and, optionally, selection, the cells aretransferred to an isolation or substantial isolation, editing, andgrowth module 940, where the cells are diluted and compartmentalizedsuch that there is an average of one cell per compartment. Oncesubstantially or largely isolated, the cells are allowed to grow for apre-determined number of doublings. Once these initial colonies areestablished, editing proceeds and the edited cells are grown toestablish colonies, which are grown to terminal size (e.g., the coloniesare normalized). In some embodiments, editing is induced by one or moreof the editing components, preferably the gRNA, being under the controlof an inducible promoter. In some embodiments, the inducible promoter isactivated by a rise in temperature and “deactivated” by lowering thetemperature. Similarly, in embodiments where the solid wall devicecomprises a filter forming the bottom of the microwell, the solid walldevice can be transferred to a plate (e.g., an agar plate or even toliquid medium) comprising a medium with a component that activates orinduces editing, then transferred to a medium that deactivates editing.In solid wall devices such as those described herein, induction ofediting and deactivation of editing can take place by media exchange.Once the colonies are grown to terminal size, the colonies are pooled.Again, isolation or substantial isolation overcomes growth bias fromunedited cells and growth bias resulting from fitness effects ofdifferent edits.

The recovery, selection, and isolation/incubation/editing andnormalization modules may all be separate, may be arranged and combinedas shown in FIG. 9, or may be arranged or combined in otherconfigurations. In certain embodiments, all of recovery, selection,isolation or substantial isolation, growth (e.g., incubation), editing,and normalization are performed in a solid wall device described inrelation to FIGS. 3A-3E and 4A-4Y. Alternatively, recovery, selection,and dilution, if necessary, are performed in liquid medium in a separatevessel such as in a rotating growth vial (module), then transferred tothe isolation/incubation/editing and normalization module.

Once the normalized cell colonies are pooled, and the cells may bestored, e.g., in a storage unit or module 912, where the cells can bekept at, e.g., 4° C. until the cells are retrieved for further study914. Alternatively, the cells may be used in another round of editing.The multi-module cell processing instrument 900 is controlled by aprocessor 942 configured to operate the instrument based on user input,as directed by one or more scripts, or as a combination of user input ora script. The processor 942 may control the timing, duration,temperature, and operations of the various modules of the instrument 900and the dispensing of reagents. For example, the processor 942 may coolthe cells post-transformation until editing is desired, upon which timethe temperature may be raised to a temperature conducive of genomeediting and cell growth. The processor may be programmed with standardprotocol parameters from which a user may select, a user may specify oneor more parameters manually or one or more scripts associated with thereagent cartridge may specify one or more operations and/or reactionparameters. In addition, the processor may notify the user (e.g., via anapplication to a smart phone or other device) that the cells havereached the target OD as well as update the user as to the progress ofthe cells in the various modules in the multi-module system.

The automated multi-module cell processing instrument 900 is anuclease-directed genome editing system and can be used in singleediting systems (e.g., introducing one or more edits to a cellulargenome in a single editing process). The system of FIG. 10, describedbelow, is configured to perform sequential editing, e.g., usingdifferent nuclease-directed systems sequentially to provide two or moregenome edits in a cell; and/or recursive editing, e.g. utilizing asingle nuclease-directed system to introduce sequentially two or moregenome edits in a cell.

FIG. 10 illustrates another embodiment of an automated multi-module cellprocessing instrument configured to perform isolation or substantialisolation of cells, growth, incubation, editing and normalization ofcell colonies. This embodiment depicts an exemplary system that performsrecursive gene editing on a cell population. As with the embodimentshown in FIG. 9, the cell processing instrument 1000 may include ahousing 1044, a reservoir for storing cells to be transformed ortransfected 1002, and a cell growth module (comprising, e.g., a rotatinggrowth vial) 1004. The cells to be transformed are transferred from areservoir to the cell growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing or transfer the cells to a cellconcentration module 1060 where the cells are subjected to bufferexchange and rendered electrocompetent, and the volume of the cells maybe reduced substantially. Once the cells have been concentrated to anappropriate volume, the cells are transferred to electroporation device1008. In addition to the reservoir for storing cells, the multi-modulecell processing instrument 1000 includes a reservoir for storing thevector pre-assembled with editing oligonucleotide cassettes 1052. Thepre-assembled nucleic acid vectors are transferred to theelectroporation device 1008, which already contains the cell culturegrown to a target OD. In the electroporation device 1008, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into an optional recovery module 1056, where thecells are allowed to recover briefly post-transformation.

After recovery, the cells may be transferred to a storage unit or module1012, where the cells can be stored at, e.g., 4° C. for laterprocessing, or the cells may be diluted and transferred to a solid wallselection, isolation or substantial isolation/incubation/editing andnormalization module 1058. In the multi-process module 1058, the cellsare arrayed such that there is an average of one cell per microwell. Thearrayed cells may be in selection medium to select for cells that havebeen transformed or transfected with the editing vector(s). Oncesubstantially or largely isolated, the cells grow through 2-50 doublingsand establish colonies. Once colonies are established, editing proceeds.As described above, in some embodiments the gRNA and other editingcomponents are under the control of an inducible promoter, and editingis induced by providing conditions (e.g., temperature, addition of aninducing or repressing chemical) to induce editing. Once editing isallowed to proceed, the cells are allowed to grow to terminal size(e.g., normalization of the colonies) in the microwells and then can beflushed out of the microwells and pooled, then transferred to the cellretrieval unit 1014 or can be transferred back to a growth module 1004for another round of editing. In between pooling and transfer to agrowth module, there may be one or more additional steps, such as cellrecovery, medium exchange, cell concentration, etc., by, e.g.,tangential flow filtration. Note that the selection/isolation orsubstantial isolation/incubation/editing and normalization modules maybe the same module, where all processes are performed in the solid walldevice, or selection and/or dilution may take place in a separate vesselbefore the cells are transferred to the solid wall isolation orsubstantial isolation/incubation/editing and normalization module (SWIINmodule). Once the putatively-edited cells are pooled, they may besubjected to another round of editing, beginning with growth, cellconcentration and treatment to render electrocompetent, andtransformation by yet another donor nucleic acid in another editingcassette via the electroporation module 1008.

In electroporation device 1008, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., editing cassettes.The multi-module cell processing instrument 1000 exemplified in FIG. 10is controlled by a processor 1042 configured to operate the instrumentbased on user input or is controlled by one or more scripts including atleast one script associated with the reagent cartridge. The processor1042 may control the timing, duration, and temperature of variousprocesses, the dispensing of reagents, and other operations of thevarious modules of the automated multi-module cell processing instrument1000. For example, a script or the processor may control the dispensingof cells, reagents, vectors, and editing oligonucleotides; which editingoligonucleotides are used for cell editing and in what order; the time,temperature and other conditions used in the recovery and expressionmodule; the wavelength at which OD is read in the cell growth module,the target OD to which the cells are grown, and the target time at whichthe cells will reach the target OD. In addition, the processor may beprogrammed to notify a user (e.g., via an application to a smart phoneor other device) as to the progress of the cells in the automatedmulti-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 10, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is to say that double-edited cells AB may be combined with andedited by vectors X, Y, and Z to produce triple-edited edited cells ABX,ABY, and ABZ; double-edited cells AC may be combined with and edited byvectors X, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ;and double-edited cells AD may be combined with and edited by vectors X,Y, and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine+editing vector in a singlevector system). “Curing” is a process in which one or more vectors usedin the prior round of editing is eliminated from the transformed cells.Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined vector) nonfunctional; diluting the vector(s) in thecell population via cell growth (that is, the more growth cycles thecells go through, the fewer daughter cells will retain the editing orengine vector(s)), or by, e.g., utilizing a heat-sensitive origin ofreplication on the editing or engine vector (or combined engine+editingvector). The conditions for curing will depend on the mechanism used forcuring; that is, in this example, how the curing plasmid cleaves theediting and/or engine plasmid. For details of curing protocols useful inthe present methods, modules and instruments, see U.S. Ser. No.62/857,967, filed 6 Jun. 2019.

Production of Cell Libraries Using Automated Editing Methods, Modules,and Instruments

In one aspect, the present disclosure provides automated editingmethods, modules, instruments, and automated multi-module cell editinginstruments for creating a library of cells that vary the expression,levels and/or activity of RNAs and/or proteins of interest in variouscell types using various editing strategies, as described herein in moredetail. Accordingly, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure. These celllibraries may have different targeted edits, including but not limitedto gene knockouts, gene knock-ins, insertions, deletions, singlenucleotide edits, short tandem repeat edits, frameshifts, triplet codonexpansion, and the like in cells of various organisms. These edits canbe directed to coding or non-coding regions of the genome and arepreferably rationally designed.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells that vary DNA-linked processes. For example, the celllibrary may include individual cells having edits in DNA binding sitesto interfere with DNA binding of regulatory elements that modulateexpression of selected genes. In addition, cell libraries may includeedits in genomic DNA that impact on cellular processes such asheterochromatin formation, switch-class recombination and VDJrecombination.

In specific aspects, the cell libraries are created using multiplexedediting of individual cells within a cell population, with multiplecells within a cell population are edited in a single round of editing,i.e., multiple changes within the cells of the cell library are in asingle automated operation. The libraries that can be created in asingle multiplexed automated operation can comprise as many as 500edited cells, 1000 edited cells, 2000 edited cells, 5000 edited cells,10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 editedcells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells,900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells,3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells,6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells,9,000,000 edited cells, 10,000,000 edited cells or more.

In other specific aspects, the cell libraries are created usingrecursive editing of individual cells within a cell population, withedits being added to the individual cells in two or more rounds ofediting. The use of recursive editing results in the amalgamation of twoor more edits targeting two or more sites in the genome in individualcells of the library. The libraries that can be created in an automatedrecursive operation can comprise as many as 500 edited cells, 1000edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells,50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 editedcells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells,1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells,4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells,7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells,10,000,000 edited cells or more.

Examples of non-automated editing strategies that can be modified basedon the present specification to utilize the automated systems can befound, e.g., U.S. Pat. Nos. 8,110,360, 8,332,160, 9,988,624,20170316353, and 20120277120.

In specific aspects, recursive editing can be used to first create acell phenotype, and then later rounds of editing used to reverse thephenotype and/or accelerate other cell properties.

In some aspects, the cell library comprises edits for the creation ofunnatural amino acids in a cell.

In specific aspects, the disclosure provides edited cell librarieshaving edits in one or more regulatory elements created using theautomated editing methods, automated multi-module cell editinginstruments of the disclosure. The term “regulatory element” refers tonucleic acid molecules that can influence the transcription and/ortranslation of an operably linked coding sequence in a particularenvironment and/or context. This term is intended to include allelements that promote or regulate transcription, and RNA stabilityincluding promoters, core elements required for basic interaction of RNApolymerase and transcription factors, upstream elements, enhancers, andresponse elements (see, e.g., Lewin, “Genes V” (Oxford University Press,Oxford) pages 847-873). Exemplary regulatory elements in prokaryotesinclude, but are not limited to, promoters, operator sequences and aribosome binding sites. Regulatory elements that are used in eukaryoticcells may include, but are not limited to, promoters, enhancers,insulators, splicing signals and polyadenylation signals.

Preferably, the edited cell library includes rationally designed editsthat are designed based on predictions of protein structure, expressionand/or activity in a particular cell type. For example, rational designmay be based on a system-wide biophysical model of genome editing with aparticular nuclease and gene regulation to predict how different editingparameters including nuclease expression and/or binding, growthconditions, and other experimental conditions collectively control thedynamics of nuclease editing. See, e.g., Farasat and Salis, PLoS ComputBiol., 29:12(1):e1004724 (2016).

In one aspect, the present disclosure provides the creation of a libraryof edited cells with various rationally designed regulatory sequencescreated using the automated editing instrumentation, systems and methodsof the invention. For example, the edited cell library can includeprokaryotic cell populations created using set of constitutive and/orinducible promoters, enhancer sequences, operator sequences and/orribosome binding sites. In another example, the edited cell library caninclude eukaryotic sequences created using a set of constitutive and/orinducible promoters, enhancer sequences, operator sequences, and/ordifferent Kozak sequences for expression of proteins of interest.

In some aspects, the disclosure provides cell libraries including cellswith rationally designed edits comprising one or more classes of editsin sequences of interest across the genome of an organism. In specificaspects, the disclosure provides cell libraries including cells withrationally designed edits comprising one or more classes of edits insequences of interest across a subset of the genome. For example, thecell library may include cells with rationally designed edits comprisingone or more classes of edits in sequences of interest across the exome,e.g., every or most every open reading frame of the genome. For example,the cell library may include cells with rationally designed editscomprising one or more classes of edits in sequences of interest acrossthe kinome. In yet another example, the cell library may include cellswith rationally designed edits comprising one or more classes of editsin sequences of interest across the secretome. In yet other aspects, thecell library may include cells with rationally designed edits created toanalyze various isoforms of proteins encoded within the exome, and thecell libraries can be designed to control expression of one or morespecific isoforms, e.g., for transcriptome analysis.

Importantly, in certain aspects the cell libraries may comprise editsusing randomized sequences, e.g., randomized promoter sequences, toreduce similarity between expression of one or more proteins inindividual cells within the library. Additionally, the promoters in thecell library can be constitutive, inducible or both to enable strongand/or titratable expression.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells comprising edits to identify optimum expression of aselected gene target. For example, production of biochemicals throughmetabolic engineering often requires the expression of pathway enzymes,and the best production yields are not always achieved by the highestamount of the target pathway enzymes in the cell, but rather byfine-tuning of the expression levels of the individual enzymes andrelated regulatory proteins and/or pathways. Similarly, expressionlevels of heterologous proteins sometimes can be experimentally adjustedfor optimal yields.

The most obvious way that transcription impacts on gene expressionlevels is through the rate of Pol II initiation, which can be modulatedby combinations of promoter or enhancer strength and trans-activatingfactors (Kadonaga, et al., Cell, 116(2):247-57 (2004)). In eukaryotes,elongation rate may also determine gene expression patterns byinfluencing alternative splicing (Cramer et al., PNAS USA,94(21):11456-60 (1997)). Failed termination on a gene can impair theexpression of downstream genes by reducing the accessibility of thepromoter to Pol II (Greger, et al., PNAS USA, 97(15):8415-20 (2000)).This process, known as transcriptional interference, is particularlyrelevant in lower eukaryotes, as they often have closely spaced genes.

In some embodiments the present disclosure provides methods foroptimizing cellular gene transcription. Gene transcription is the resultof several distinct biological phenomena, including transcriptionalinitiation (RNAp recruitment and transcriptional complex formation),elongation (strand synthesis/extension), and transcriptional termination(RNAp detachment and termination).

Site-Directed Mutagenesis

Cell libraries can be created using the automated editing methods,modules, instruments, and systems employing site-directed mutagenesis,i.e., when the amino acid sequence of a protein or other genomic featuremay be altered by deliberately and precisely by mutating the protein orgenomic feature. These cell lines can be useful for various purposes,e.g., for determining protein function within cells, the identificationof enzymatic active sites within cells, and the design of novelproteins. For example, site-directed mutagenesis can be used in amultiplexed fashion to exchange a single amino acid in the sequence of aprotein for another amino acid with different chemical properties. Thisallows one to determine the effect of a rationally designed or randomlygenerated mutation in individual cells within a cell population. See,e.g., Berg, et al. Biochemistry, Sixth Ed. (New York: W.H. Freeman andCompany) (2007).

In another example, edits can be made to individual cells within a celllibrary to substitute amino acids in binding sites, such as substitutionof one or more amino acids in a protein binding site for interactionwithin a protein complex or substitution of one or more amino acids inenzymatic pockets that can accommodate a cofactor or ligand. This classof edits allows the creation of specific manipulations to a protein tomeasure certain properties of one or more proteins, includinginteraction with other cofactors, ligands, etc. within a proteincomplex.

In yet another examples, various edit types can be made to individualcells within a cell library using site specific mutagenesis for studyingexpression quantitative trait loci (eQTLs). An eQTL is a locus thatexplains a fraction of the genetic variance of a gene expressionphenotype. The libraries of the invention would be useful to evaluateand link eQTLs to actual diseased states.

In specific aspects, the edits introduced into the cell libraries of thedisclosure may be created using rational design based on known orpredicted structures of proteins. See, e.g., Chronopoulou and Labrou,Curr Protoc Protein Sci.; Chapter 26:Unit 26.6 (2011). Suchsite-directed mutagenesis can provide individual cells within a librarywith one or more site-directed edits, and preferably two or moresite-directed edits (e.g., combinatorial edits) within a cellpopulation.

In other aspects, cell libraries of the disclosure are created usingsite-directed codon mutation “scanning” of all or substantially all ofthe codons in the coding region of a gene. In this fashion, individualedits of specific codons can be examined for loss-of-function orgain-of-function based on specific polymorphisms in one or more codonsof the gene. These libraries can be a powerful tool for determiningwhich genetic changes are silent or causal of a specific phenotype in acell or cell population. The edits of the codons may be randomlygenerated or may be rationally designed based on known polymorphismsand/or mutations that have been identified in the gene to be analyzed.Moreover, using these techniques on two or more genes in a single in apathway in a cell may determine potential protein:protein interactionsor redundancies in cell functions or pathways.

For example, alanine scanning can be used to determine the contributionof a specific residue to the stability or function of given protein.See, e.g., Lefèvre, et al., Nucleic Acids Research, Volume 25(2):447-448(1997). Alanine is often used in this codon scanning technique becauseof its non-bulky, chemically inert, methyl functional group that canmimic the secondary structure preferences that many of the other aminoacids possess. Codon scanning can also be used to determine whether theside chain of a specific residue plays a significant role in cellfunction and/or activity. Sometimes other amino acids such as valine orleucine can be used in the creation of codon scanning cell libraries ifconservation of the size of mutated residues is needed.

In other specific aspects, cell libraries can be created using theautomated editing methods, automated multi-module cell editinginstruments of the invention to determine the active site of a proteinsuch as an enzyme or hormone, and to elucidate the mechanism of actionof one or more of these proteins in a cell library. Site-directedmutagenesis associated with molecular modeling studies can be used todiscover the active site structure of an enzyme and consequently itsmechanism of action. Analysis of these cell libraries can provide anunderstanding of the role exerted by specific amino acid residues at theactive sites of proteins, in the contacts between subunits of proteincomplexes, on intracellular trafficking and protein stability/half-lifein various genetic backgrounds.

Saturation Mutagenesis

In some aspects, the cell libraries created using the automated editingmethods and automated multi-module cell editing instruments of thedisclosure may be saturation mutagenesis libraries, in which a singlecodon or set of codons is randomized to produce all possible amino acidsat the position of a particular gene or genes of interest. These celllibraries can be particularly useful to generate variants, e.g., fordirected evolution. See, e.g., Chica, et al., Current Opinion inBiotechnology 16 (4): 378-384 (2005); and Shivange, Current Opinion inChemical Biology, 13 (1): 19-25 (2009).

In some aspects, edits comprising different degenerate codons can beused to encode sets of amino acids in the individual cells in thelibraries. Because some amino acids are encoded by more codons thanothers, the exact ratio of amino acids cannot be equal. In certainaspects, more restricted degenerate codons are used. ‘NNK’ and ‘NNS’have the benefit of encoding all 20 amino acids, but still encode a stopcodon 3% of the time. Alternative codons such as ‘NDT’, ‘DBK’ avoid stopcodons entirely, and encode a minimal set of amino acids that stillencompass all the main biophysical types (anionic, cationic, aliphatichydrophobic, aromatic hydrophobic, hydrophilic, small).

In specific aspects, the non-redundant saturation mutagenesis, in whichthe most commonly used codon for a particular organism is used in thesaturation mutagenesis editing process.

Promoter Swaps and Ladders

One mechanism for analyzing and/or optimizing expression of one or moregenes of interest is through the creation of a “promoter swap” celllibrary, in which the cells comprise genetic edits that have specificpromoters linked to one or more genes of interest. Accordingly, the celllibraries created using the methods, automated multi-module cell editinginstruments of the disclosure may be promoter swap cell libraries, whichcan be used, e.g., to increase or decrease expression of a gene ofinterest to optimize a metabolic or genetic pathway. In some aspects,the promoter swap cell library can be used to identify an increase orreduction in the expression of a gene that affects cell vitality orviability, e.g., a gene encoding a protein that impacts on the growthrate or overall health of the cells. In some aspects, the promoter swapcell library can be used to create cells having dependencies and logicbetween the promoters to create synthetic gene networks. In someaspects, the promoter swaps can be used to control cell to cellcommunication between cells of both homogeneous and heterogeneous(complex tissues) populations in nature.

The cell libraries can utilize any given number of promoters that havebeen grouped together based upon exhibition of a range of expressionstrengths and any given number of target genes. The ladder of promotersequences vary expression of at least one locus under at least onecondition. This ladder is then systematically applied to a group ofgenes in the organism using the automated editing methods, automatedmulti-module cell editing instruments of the disclosure.

In specific aspects, the cell library formed using the automated editingprocesses, modules and systems of the disclosure include individualcells that are representative of a given promoter operably linked to oneor more target genes of interest in an otherwise identical geneticbackground. Examples of non-automated editing strategies that can bemodified to utilize the automated systems can be found, e.g., in U.S.Pat. No. 9,988,624.

In specific aspects, the promoter swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of promoters that act as a “promoter ladder” for expression of thegenes of interest. For example, the cells are edited so that one or moreindividual genes of interest are edited to be operably linked with thedifferent promoters in the promoter ladder. When an endogenous promoterdoes not exist, its sequence is unknown, or it has been previouslychanged in some manner, the individual promoters of the promoter laddercan be inserted in front of the genes of interest. These produced celllibraries have individual cells with an individual promoter of theladder operably linked to one or more target genes in an otherwiseidentical genetic context.

The promoters are generally selected to result in variable expressionacross different loci, and may include inducible promoters, constitutivepromoters, or both.

The set of target genes edited using the promoter ladder can include allor most open reading frames (ORFs) in a genome, or a selected subset ofthe genome, e.g., the ORFs of the kinome or a secretome. In someaspects, the target genes can include coding regions for variousisoforms of the genes, and the cell libraries can be designed toexpression of one or more specific isoforms, e.g., for transcriptomeanalysis using various promoters.

The set of target genes can also be genes known or suspected to beinvolved in a particular cellular pathway, e.g. a regulatory pathway orsignaling pathway. The set of target genes can be ORFs related tofunction, by relation to previously demonstrated beneficial edits(previous promoter swaps or previous SNP swaps), by algorithmicselection based on epistatic interactions between previously generatededits, other selection criteria based on hypotheses regarding beneficialORF to target, or through random selection. In specific embodiments, thetarget genes can comprise non-protein coding genes, including non-codingRNAs.

Editing of other functional genetic elements, including insulatorelements and other genomic organization elements, can also be used tosystematically vary the expression level of a set of target genes, andcan be introduced using the methods, automated multi-module cell editinginstruments of the disclosure. In one aspect, a population of cells isedited using a ladder of enhancer sequences, either alone or incombination with selected promoters or a promoter ladder, to create acell library having various edits in these enhancer elements. In anotheraspect, a population of cells is edited using a ladder of ribosomebinding sequences, either alone or in combination with selectedpromoters or a promoter ladder, to create a cell library having variousedits in these ribosome binding sequences.

In another aspect, a population of cells is edited to allow theattachment of various mRNA and/or protein stabilizing or destabilizingsequences to the 5′ or 3′ end, or at any other location, of a transcriptor protein.

In certain aspects, a population of cells of a previously establishedcell line may be edited using the automated editing methods, modules,instruments, and systems of the disclosure to create a cell library toimprove the function, health and/or viability of the cells. For example,many industrial strains currently used for large scale manufacturinghave been developed using random mutagenesis processes iteratively overa period of many years, sometimes decades. Unwanted neutral anddetrimental mutations were introduced into strains along with beneficialchanges, and over time this resulted in strains with deficiencies inoverall robustness and key traits such as growth rates. In anotherexample, mammalian cell lines continue to mutate through the passage ofthe cells over periods of time, and likewise these cell lines can becomeunstable and acquire traits that are undesirable. The automated editingmethods, automated multi-module cell editing instruments of thedisclosure can use editing strategies such as SNP and/or STR swapping,indel creation, or other techniques to remove or change the undesirablegenome sequences and/or introducing new genome sequences to address thedeficiencies while retaining the desirable properties of the cells.

When recursive editing is used, the editing in the individual cells inthe edited cell library can incorporate the inclusion of “landing pads”in an ectopic site in the genome (e.g., a CarT locus) to optimizeexpression, stability and/or control.

In some embodiments, each library produced having individual cellscomprising one or more edits (either introducing or removing) iscultured and analyzed under one or more criteria (e.g., production of achemical or product of interest). The cells possessing the specificcriteria are then associated, or correlated, with one or more particularedits in the cell. In this manner, the effect of a given edit on anynumber of genetic or phenotypic traits of interest can be determined.The identification of multiple edits associated with particular criteriaor enhanced functionality/robustness may lead to cells with highlydesirable characteristics.

Knock-Out or Knock-in Libraries

In certain aspects, the present disclosure provides automated editingmethods, modules, instruments and systems for creating a library ofcells having “knock-out” (KO) or “knock-in” (KI) edits of various genesof interest. Thus, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure that have one ormore mutations that remove or reduce the expression of selected genes ofinterest to interrogate the effect of these edits on gene function inindividual cells within the cell library.

The cell libraries can be created using targeted gene KO (e.g., viainsertion/deletion) or KOs (e.g., via homologous directed repair). Forexample, double strand breaks are often repaired via the non-homologousend joining DNA repair pathway. The repair is known to be error prone,and thus insertions and deletions may be introduced that can disruptgene function. Preferably the edits are rationally designed tospecifically affect the genes of interest, and individual cells can becreated having a KI or KI of one or more locus of interest. Cells havinga KO or KI of two or more loci of interest can be created usingautomated recursive editing of the disclosure.

In specific aspects, the KO or KI cell libraries are created usingsimultaneous multiplexed editing of cells within a cell population, andmultiple cells within a cell population are edited in a single round ofediting, i.e., multiple changes within the cells of the cell library arein a single automated operation. In other specific aspects, the celllibraries are created using recursive editing of individual cells withina cell population, and results in the amalgamation of multiple edits oftwo or more sites in the genome into single cells.

SNP or Short Tandem Repeat Swaps

In one aspect, cell libraries are created using the automated editingmethods, automated multi-module cell editing instruments of thedisclosure by systematic introducing or substituting single nucleotidepolymorphisms (“SNPs”) into the genomes of the individual cells tocreate a “SNP swap” cell library. In some embodiments, the SNP swappingmethods of the present disclosure include both the addition ofbeneficial SNPs, and removing detrimental and/or neutral SNPs. The SNPswaps may target coding sequences, non-coding sequences, or both.

In another aspect, a cell library is created using the automated editingmethods, modules, instruments, instruments, and systems of thedisclosure by systematic introducing or substituting short tandemrepeats (“STR”) into the genomes of the individual cells to create an“STR swap” cell library. In some embodiments, the STR swapping methodsof the present disclosure include both the addition of beneficial STRs,and removing detrimental and/or neutral STRs. The STR swaps may targetcoding sequences, non-coding sequences, or both.

In some embodiments, the SNP and/or STR swapping used to create the celllibrary is multiplexed, and multiple cells within a cell population areedited in a single round of editing, i.e., multiple changes within thecells of the cell library are in a single automated operation. In otherembodiments, the SNP and/or STR swapping used to create the cell libraryis recursive, and results in the amalgamation of multiple beneficialsequences and/or the removal of detrimental sequences into single cells.Multiple changes can be either a specific set of defined changes or apartly randomized, combinatorial library of mutations. Removal ofdetrimental mutations and consolidation of beneficial mutations canprovide immediate improvements in various cellular processes. Removal ofgenetic burden or consolidation of beneficial changes into a strain withno genetic burden also provides a new, robust starting point foradditional random mutagenesis that may enable further improvements.

SNP swapping overcomes fundamental limitations of random mutagenesisapproaches as it is not a random approach, but rather the systematicintroduction or removal of individual mutations across cells.

Splice Site Editing

RNA splicing is the process during which introns are excised and exonsare spliced together to create the mRNA that is translated into aprotein. The precise recognition of splicing signals by cellularmachinery is critical to this process. Accordingly, in some aspects, apopulation of cells is edited using a systematic editing to known and/orpredicted splice donor and/or acceptor sites in various loci to create alibrary of splice site variants of various genes. Such editing can helpto elucidate the biological relevance of various isoforms of genes in acellular context. Sequences for rational design of splicing sites ofvarious coding regions, including actual or predicted mutationsassociated with various mammalian disorders, can be predicted usinganalysis techniques such as those found in Nalla and Rogan, Hum Mutat,25:334-342 (2005); Divina, et al., Eur J Hum Genet, 17:759-765 (2009);Desmet, et el., Nucleic Acids Res, 37:e67 (2009); Faber, et al., BMCBioinformatics, 12(suppl 4):S2 (2011).

Start/Stop Codon Exchanges and Incorporation of Nucleic Acid Analogs

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of the disclosure, where the libraries are created byswapping start and stop codon variants throughout the genome of anorganism or for a selected subset of coding regions in the genome, e.g.,the kinome or secretome. In the cell library, individual cells will haveone or more start or stop codons replacing the native start or stopcodon for one or more gene of interest.

For example, typical start codons used by eukaryotes are ATG (AUG) andprokaryotes use ATG (AUG) the most, followed by GTG (GUG) and TTG (UUG).The cell library may include individual cells having substitutions forthe native start codons for one or more genes of interest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing ATG start codons with TTG in front ofselected genes of interest. In other aspects, the present disclosureprovides for automated creation of a cell library by replacing ATG startcodons with GTG. In other aspects, the present disclosure provides forautomated creation of a cell library by replacing GTG start codons withATG. In other aspects, the present disclosure provides for automatedcreation of a cell library by replacing GTG start codons with TTG. Inother aspects, the present disclosure provides for automated creation ofa cell library by replacing TTG start codons with ATG. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TTG start codons with GTG.

In other examples, typical stop codons for S. cerevisiae and mammals areTAA (UAA) and TGA (UGA), respectively. The typical stop codon formonocotyledonous plants is TGA (UGA), whereas insects and E. colicommonly use TAA (UAA) as the stop codon (Dalphin. et al., Nucl. AcidsRes., 24: 216-218 (1996)). The cell library may include individual cellshaving substitutions for the native stop codons for one or more genes ofinterest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing TAA stop codons with TAG. In otheraspects, the present disclosure provides for automated creation of acell library by replacing TAA stop codons with TGA. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TGA stop codons with TAA. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TGA stop codons with TAG. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TAG stop codons with TAA. In other aspects, the presentinvention teaches automated creation of a cell library by replacing TAGstop codons with TGA.

Terminator Swaps and Ladders

One mechanism for identifying optimum termination of a pre-spliced mRNAof one or more genes of interest is through the creation of a“terminator swap” cell library, in which the cells comprise geneticedits that have specific terminator sequences linked to one or moregenes of interest. Accordingly, the cell libraries created using themethods, modules, instruments and systems of the disclosure may beterminator swap cell libraries, which can be used, e.g., to affect mRNAstability by releasing transcripts from sites of synthesis. In otherembodiments, the terminator swap cell library can be used to identify anincrease or reduction in the efficiency of transcriptional terminationand thus accumulation of unspliced pre-mRNA (e.g., West and Proudfoot,Mol Cell; 33(3-9); 354-364 (2009)) and/or 3′ end processing (e.g., West,et al., Mol Cell. 29(5):600-10 (2008)). In the case where a gene islinked to multiple termination sites, the edits may edit a combinationof edits to multiple terminators that are associated with a gene.Additional amino acids may also be added to the ends of proteins todetermine the effect on the protein length on terminators.

The cell libraries can utilize any given number of edits of terminatorsthat have been selected for the terminator ladder based upon exhibitionof a range of activity and any given number of target genes. The ladderof terminator sequences vary expression of at least one locus under atleast one condition. This ladder is then systematically applied to agroup of genes in the organism using the automated editing methods,modules, instruments and systems of the disclosure.

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of disclosure, where the libraries are created to editterminator signals in one or more regions in the genome in theindividual cells of the library. Transcriptional termination ineukaryotes operates through terminator signals that are recognized byprotein factors associated with the RNA polymerase II. For example, thecell library may contain individual eukaryotic cells with edits in genesencoding polyadenylation specificity factor (CPSF) and cleavagestimulation factor (CstF) and or gene encoding proteins recruited byCPSF and CstF factors to termination sites. In prokaryotes, twoprincipal mechanisms, termed Rho-independent and Rho-dependenttermination, mediate transcriptional termination. For example, the celllibrary may contain individual prokaryotic cells with edits in genesencoding proteins that affect the binding, efficiency and/or activity ofthese termination pathways.

In certain aspects, the present disclosure provides methods of selectingtermination sequences (“terminators”) with optimal properties. Forexample, in some embodiments, the present disclosure teaches providesmethods for introducing and/or editing one or more terminators and/orgenerating variants of one or more terminators within a host cell, whichexhibit a range of activity. A particular combination of terminators canbe grouped together as a terminator ladder, and cell libraries of thedisclosure include individual cells that are representative ofterminators operably linked to one or more target genes of interest inan otherwise identical genetic background. Examples of non-automatedediting strategies that can be modified to utilize the automatedinstruments can be found, e.g., in U.S. Pat. No. 9,988,624 to Serber etal., entitled “Microbial strain improvement by a HTP genomic engineeringplatform.”

In specific aspects, the terminator swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of terminators that act as a “terminator ladder” for expression ofthe genes of interest. For example, the cells are edited so that theendogenous promoter is operably linked to the individual genes ofinterest are edited with the different promoters in the promoter ladder.When the endogenous promoter does not exist, its sequence is unknown, orit has been previously changed in some manner, the individual promotersof the promoter ladder can be inserted in front of the genes ofinterest. These produced cell libraries have individual cells with anindividual promoter of the ladder operably linked to one or more targetgenes in an otherwise identical genetic context. The terminator ladderin question is then associated with a given gene of interest.

The terminator ladder can be used to more generally affect terminationof all or most ORFs in a genome, or a selected subset of the genome,e.g., the ORFs of a kinome or a secretome. The set of target genes canalso be genes known or suspected to be involved in a particular cellularpathway, e.g. a regulatory pathway or signaling pathway. The set oftarget genes can be ORFs related to function, by relation to previouslydemonstrated beneficial edits (previous promoter swaps or previous SNPswaps), by algorithmic selection based on epistatic interactions betweenpreviously generated edits, other selection criteria based on hypothesesregarding beneficial ORF to target, or through random selection. Inspecific embodiments, the target genes can comprise non-protein codinggenes, including non-coding RNAs.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Other equivalent methods, steps and compositionsare intended to be included in the scope of the invention. Efforts havebeen made to ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Celsius, and pressure is at or near atmospheric.

Example 1: Assessing the Fitness of a Model System

Basic components of a model system were assessed. The model systemcomprised E. coli cells transformed with an engine vector, where theengine vector comprised a coding sequence for a MAD nuclease (i.e., MAD4 or MAD 7 nuclease) under the control of the pL temperature induciblepromoter, a chloramphenicol resistance marker, and the λ Redrecombineering system under the control of the pBAD promoter (induced byaddition of arabinose to the growth medium). The E. coli cells were alsotransformed with an editing vector comprising an editingoligonucleotide, which in this model system was a library of editingoligonucleotides each configured to inactivate galK, where successfulediting results in white (versus red) colonies when plated on MacConkeyagar supplemented with galactose as the sole carbon source. In addition,the editing vector comprised a gRNA coding sequence under the control ofthe pL temperature inducible promoter, a carbenicillin resistancemarker, and a sequence to remove, mutate, or otherwise render inactivethe PAM region in the target sequence. FIGS. 11A and 11B depict anexemplary engine vector (FIG. 11A) and editing vector (FIG. 11B) thatmay be employed in the exemplary editing workflows described herein. InFIG. 11A, the exemplary engine vector 1110 (p197) comprises an origin ofreplication 1112, and a promoter 1114 driving expression of the genecoding for the c1857 repressor 1116 which regulates the pL promoter. Afirst pL promoter in this exemplary embodiment drives the transcriptionof the gRNA on the editing vector as described in relation to FIG. 11Bbelow, and a second pL promoter 1118 on the engine vector drivesexpression of the nuclease 1120. The promoter driving nuclease 1120 maybe an inducible or a constitutive promoter; however, the tightestregulation of the nucleic acid-guided nuclease system is achieved byusing an inducible promoter to drive expression of the nuclease as wellas the guide nucleic acid. Again, like inducible promoters and differentinducible promoters may be used to drive the transcription of the guidenucleic acid and the nuclease. The pL promoter can be regulated (e.g.,repressed or induced) by the thermolabile c1857 repressor 1116 which isactive at permissive temperatures (e.g., 30° C.) and inactive at highertemperatures (e.g., 42° C.). The regulation of the pL promoter is shownin more detail in relation to FIG. 1C above.

The engine vector in FIG. 11A further comprises a pBAD promoter 1140driving expression of the components of the λ Red recombineering system1142. The pBAD promoter, like the pL promoter, is an inducible promoter,where the pBAD promoter is regulated (induced) by the addition ofarabinose to the growth medium. Note that in this exemplary bacterialediting system, a recombineering system such as the λ Red recombineeringsystem is provided as a component of the nucleic acid-guided nucleaseediting system to repair the DNA breaks that occur during editing. Insome embodiments, however, the cells to be edited may already comprise arecombineering system (e.g., episomally, integrated into the cellulargenome, or naturally). Also, although the λ Red recombineering system isexemplified here, it should be understood that other recombineeringsystems may be employed. Further, cells—such as yeast, plant and animalcells—do not require a recombineering system equivalent to the λ Redrecombineering system to repair the DNA breaks that result from editing.Thus the nucleic acid-guided nuclease editing components for, e.g.,yeast, plant and animal cells do not need to include a heterologousrecombineering system. Finally, exemplary engine vector 1110 comprises apromoter 1150 driving expression of a chloramphenicol selectable marker1152. Additionally, the engine vector and all other vectors orconstructs used in the disclosed method comprise appropriate controlelements (e.g., polyadenylation signals, enhancers) operably-linked tothe nucleic acid-guided nuclease editing system components.

FIG. 11B depicts an exemplary editing vector 1130 (pLFORGE). Editingvector 1130 comprises an origin of replication 1132, and a pL promoter1134 driving expression of an editing cassette 1136. The editingcassette comprises a coding sequence for a gRNA 1144, a spacer 1146, anda donor DNA 1158 (comprising homology arms flanking a desired edit to bemade to the target sequence). Editing vector 1130 also comprises apromoter 1154 driving expression of a carbenicillin selectable marker1156. Note that the pL inducible promoter 1134 is encoded on the vectorbackbone and is not part of the editing cassette 1136; however, in otherembodiments the inducible promoter driving transcription of the gRNA maybe part of the editing cassette. FIGS. 11A and 11B depict exemplaryengine and editing vectors, respectively, but it should be recognized byone of ordinary skill in the art given the guidance of the presentdescription that all elements of the nucleic acid-guided nucleaseediting system may be contained on a single plasmid, or the elementsshown on the engine and editing vectors of FIGS. 11A and 11B may resideon a different vector than shown. For example, the pBAD promoter and λRed recombineering system may be contained on the editing vector ratherthan the engine vector; likewise, the gene for the c1857 repressor maybe contained on the editing vector rather than the engine vector.

FIG. 12 is a bar graph showing the transformation efficiencies observedfor galK gRNA targeting cassettes under a variety of promoters.Transformation efficiency of uninduced promoters provides a proxy forpromoter leakiness. If there is high basal expression of gRNA (e.g.,“leakiness”), there will be low CFU. If there is a low basal expressionof gRNA (e.g., tight regulation), there will be high CFU. The non-targetgRNAs experiment was performed with a pool of cassettes designed totarget inactive PAM sequences that are unable to promote gRNA andnuclease binding and cleavage. For each of the promoters depicted (pL,pBAD, and sigma32) a cassette targeting galK with a TTTC PAM and aspacer configured to make a stop codon insertion at amino acid D70 wascloned downstream of the promoter. Plasmid concentrations werenormalized to 50 ng/pL and transformed in equal cell volumes. CFU fromserial dilution platings were used to derive the transformationefficiencies expressed as CFU/n. The J23119 promoter is a constitutivepromoter, the pL promoter is inducible by high temperature, the pBADpromoter is induced by the presence of arabinose in the growth medium,and the sigma32 promoter is induced in stationary phase cells. For theMAD7 nuclease, the sigma32 promoter shows leaky expression, while boththe pL and pBAD promoters are tightly repressed.

FIG. 13 is a graph showing the effects of thermal induction on cellviability. In this experiment, a pool of cassettes were designed totarget an inactive PAM sequence. Cells were diluted 1/50 v:v intomicrotiter plates containing 200 μL LB+chlor/carb. Diluted cultures wereinduced for 1 hour at the indicated temperatures before shifting back to30° C. with continuous shaking in an Infinite M Nano+ plate reader. Tocorresponds to the time of the induction step and growth was monitoredby measuring the OD600 at 10 minute intervals. Note that hightemperature (42° C.) induction has no impact on cell viability or growthrate.

FIG. 14 shows the results of pooled induction experiments using a singlesequence verified editing cassette that targets the galK locus andintroduces a stop codon at amino acid D70. Following a three-houroutgrowth, cells were either induced at 42° C. for 15 minutes orretained at 30° C. prior to serial dilution plating. CFU/mL wascalculated based on the plating volumes. For reference the dashed linein FIG. 14 shows the typical CFU obtained in an equivalent experimentusing the constitutive J23119 promoter (data not shown). As can be seen,the absence of induction (i.e., the absence of editing) enables highefficiency transformation (10-100 fold) due to the lack of toxic dsDNAbreaks.

FIG. 15 shows the growth profiles of randomly picked variants from asilent PAM mutation (SPM) library. This 500-member library targetsregions located across the entire E. coli organism and integratessynonymous mutations that have no expected fitness effects. Colonieswere picked from agar plates of uninduced transformed cells. Cells werepicked from an agar plate and grown up in 200 μL LB+chlor/carb overnightin a 96-well microtiter plate format. 10 μL of the well content of theparent microtiter plate was then transferred to two replica daughtermicrotiter plates that received either no induction (top) or gRNA andnuclease induction (via the pL inducible promoter) for 1 hour at 42° C.(bottom). The well maps show the relative OD at 6 hours. The insertsshow examples of the full growth curves for the indicated wells as areference. The replica wells represent growth observed from the samecassette design with or without gRNA induction. While the majority ofthe wells for the no-induction plate show normal growth profiles, theinduced plate shows that a large fraction of the gRNA designs are stillactive when induced, indicated by a large lag phase before the cellsreach exponential growth. That is, the actively-editing cells havereduced viability due to DNA damage such that many in the colonies dieoff, and those edited cells that do survive grow slowly to begin with asthe cellular machinery works to repair the edit. This characteristic ofedited cells can be exploited to screen for active editing in ahigh-throughput manner.

Example 2: Preparing Nucleic Acids, Transformation

Editing Cassette Preparation: 5 nM oligonucleotides synthesized on achip were amplified using Q5 polymerase in 50 μL volumes. The PCRconditions were 95° C. for 1 minute; 8 rounds of 95° C. for 30seconds/60° C. for 30 seconds/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. Following amplification, the PCR products weresubjected to SPRI cleanup, where 30 μL SPRI mix was added to the 50 μLPCR reactions and incubated for 2 minutes. The tubes were subjected to amagnetic field for 2 minutes, the liquid was removed, and the beads werewashed 2× with 80% ethanol, allowing 1 minute between washes. After thefinal wash, the beads were allowed to dry for 2 minutes, 50 μL 0.5× TEpH 8.0 was added to the tubes, and the beads were vortexed to mix. Theslurry was incubated at room temperature for 2 minutes, then subjectedto the magnetic field for 2 minutes. The eluate was removed and the DNAquantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation:

A 10-fold serial dilution series of purified backbone was performed, andeach of the diluted backbone series was amplified under the followingconditions: 95° C. for 1 minute; then 30 rounds of 95° C. for 30seconds/60° C. for 1.5 minutes/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. After amplification, the amplified backbone wassubjected to SPRI cleanup as described above in relation to thecassettes. The backbone was eluted into 100 μL ddH₂O and quantifiedbefore isothermal nucleic acid assembly.

Isothermal Nucleic Acid Assembly:

150 ng backbone DNA was combined with 100 ng cassette DNA. An equalvolume of 2× isothermal assembly master mix was added, and the reactionwas incubated for 45 minutes at 50° C. After assembly, the assembledbackbone and cassettes were subjected to SPRI cleanup, as describedabove.

Transformation with Engine Vector:

1 μL of the engine vector DNA (comprising a coding sequence for MAD7nuclease under the control of the pL inducible promoter, achloramphenicol resistance gene, and the λ Red recombineering system)was added to 50 μL EC1 strain E. coli cells. The transformed cells wereplated on LB plates with 25 μg/mL chloramphenicol (chlor) and incubatedovernight to accumulate clonal (or substantially clonal) isolates. Thenext day, a colony was picked, grown overnight in LB+25 μg/mL chlor, andglycerol stocks were prepared from the saturated overnight culture byadding 500 μL 50% glycerol to 1000 μL culture. The stocks of EC1comprising the engine vector were frozen at −80° C.

Transformation with Editing Vector:

A 1 mL aliquot of a freshly-grown overnight culture of EC1 cellstransformed with the engine vector was added to a 250 mL flaskcontaining 100 mL LB/SOB+25 μg/mL chlor medium. The cells were grown to0.4-0.7 OD, and cell growth was halted by transferring the culture toice for 10 minutes. The cells were pelleted at 8000×g in a JA-18 rotorfor 5 minutes, washed 3× with 50 mL ice cold ddH₂O or 10% glycerol andpelleted at 8000×g in JA-18 rotor for 5 minutes. The washed cells wereresuspended in 5 mL ice cold 10% glycerol and aliquoted into 200 μLportions. Optionally at this point the glycerol stocks could be storedat −80° C. for later use. 20 μL of the prepared editing vectorisothermal nucleic acid assembly reaction was added to 30 μL chilledwater along with 10 μL E cloni® (Lucigen, Middleton, Wis.) supremecompetent cells. An aliquot of the transformed cells were spot plated tocheck the transformation efficiency, where >100× coverage was requiredto continue. The transformed E cloni® cells were outgrown in 25 mLSOB+100 μg/mL carbenicillin (carb). Glycerol stocks were generated fromthe saturated culture by adding 500 μL 50% glycerol to 1000 μL saturatedovernight culture. The stocks were frozen at −80° C. This step isoptional, providing a ready stock of the cloned editing library.Alternatively, isothermal or another assembly of the editing cassettesand the vector backbone can be performed before each editing experiment.

Example 3: High Throughput Clonal Editing

The following protocols address cell growth/survival bias due to dsDNAbreaks:

Transformation:

100 ng of the editing vector cloned library or editing vector isothermalnucleic acid assembly reaction was transformed by electroporation into100 μL competent EC1 cells containing the engine vector. Theelectroporator was set to 2400 V in 2 mm cuvette. Followingtransformation, the cells were allowed to recover for 3 hours in SOBmedium. A 10-fold dilution series of recovered cells (in H₂O) was spotplated and the resulting CFU counts/dilution ratios were used tocalculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB medium+25 μg/mLchlor and grown at 30° C. overnight. Colonies were picked and grownovernight to saturation at 30° C. in 96-well microtiter plates.

Induced Cutting, Editing, and Edit Validation:

A replicator was used to transfer colonies in wells from the overnightgrowth to 200 μL fresh SOB+1% arabinose in replicate 96-well microtiterplates. The replicator transfers approximately 1-2 μL per well resultingin a 100-fold dilution for outgrowth. The microtiter plates wereincubated at 250 rpm shaking for 3-4 hours to allow the cells to reachmid-log phase. The plates were then transferred to a static incubator at42° C. and incubated for 2 hours, then placed in a 30° C. incubator for1 hour to recover. At this point, the replicate cells were ready forgenomic prep and were validated via sequencing analysis. As described inrelation to FIG. 2B, additional replica plates of fresh SOB withoutarabinose may be used to enable functional deconvolution of inactive cutdesigns from the population.

Example 4: Rearray and Pooled Editing Using Inducible gRNAs

Transformation:

100 ng of the editing vector cloned library or isothermal nucleic acidassembly reaction was transformed by electroporation into 100 μLcompetent EC1 cells containing the engine vector. The electroporator wasset to 2400 V in 2 mm cuvette. Following transformation, the cells wereallowed to recover for 3 hours in SOB medium. A 10-fold dilution seriesof recovered cells (in H₂O) was spot plated and the resulting CFUcounts/dilution ratios were used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB medium+25 μg/mLchlor and grown at 30° C. overnight. Colonies were picked and grownovernight to saturation in 1 mL SOB containing 25 μg/mL chlor and 100μg/mL carb at 30° C. in 96-well microtiter plates.

Identification of Active Cassettes and Rearray:

A replicator was used to transfer colonies in wells from the overnightgrowth to 200 μL fresh SOB without arabinose in replicate 96-wellmicrotiter plates. The replicator transfers approximately 1-2 μL perwell resulting in a 100-fold dilution for outgrowth. The replicatemicrotiter plates were incubated at 250 rpm shaking for 3-4 hours toallow the cells to reach mid-log growth. The plates were thentransferred to a static incubator at 42° C. and incubated for 2 hours,then placed in a 30° C. incubator for 1 hour to recover. The replicatorwas then used to transfer cells from the replicate microtiter plates toagar plates with LB medium+25 μg/mL chlor/100 μg/mL carb and allowed togrow overnight. Wells with active gRNAs were identified and 50 μL ofcells from these colonies from the original 96-well microtiter platewere re-arrayed into “functionally-corrected” plates (e.g., the cellsidentified as having functional gRNAs were transferred (cherry picked)into a 96-well plate).

Pooled Editing Using Pooled Cassettes Identified as Active:

100 μL of cells identified as having functional gRNAs were transferredinto 5 mL SOB medium and grown until the culture was saturated. 1%arabinose was added to the tube and the tube was incubated for 2 hoursat 30° C. Cutting/editing was induced by transferring the tube to a 42°C. shaking water bath for 2 hours. The tube was then removed from thewater bath and allowed to recover for 2 hours at 30° C. in a 250 rpmshaking incubator. A diluted culture (approximately 10⁻⁴ to 10⁻⁵) wasplated on LB medium containing 25 μg/mL chlor and 100 μg/mL carbresulting in isolated clonal colonies. Editing was assessed/validated bytargeted or whole genome sequencing.

Example 5: Error Correction: Re-Array and Cloning

Transformation:

100 ng of the editing vector cloned library or isothermal nucleic acidassembly reaction was transformed by electroporation into 100 μLcompetent EC1 cells containing the engine vector. The electroporator wasset to 2400 V in 2 mm cuvette. Following transformation, the cells wereallowed to recover for 3 hours in SOB medium. A 10-fold dilution seriesof recovered cells (in H₂O) was spot plated and the resulting CFUcounts/dilution ratios were used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB medium+25 μg/mLchlor and grown at 30° C. overnight. Colonies were picked and grownovernight to saturation in 1 mL SOB containing 25 μg/mL chlor and 100m/mL carb at 30° C. in 96-well microtiter plates.

Identification of Active Cassettes and Rearray:

A replicator was used to transfer colonies in wells from the overnightgrowth to 200 μL fresh SOB without arabinose in replicate 96-wellmicrotiter plates. The replicator transfers approximately 1-2 μL perwell resulting in a 100-fold dilution for outgrowth. The replicatemicrotiter plates were incubated at 250 rpm shaking for 3-4 hours toallow the cells to reach mid-log growth. The plates were thentransferred to a static incubator at 42° C. and incubated for 2 hours,then placed in a 30° C. incubator for 1 hour to recover. The replicatorwas then used to transfer cells from the replicate microtiter plates toplates with LB medium+25 μg/mL chlor/100 μg/mL carb and allowed to growovernight. Wells with active gRNAs were identified and 50 μL of cellsfrom these colonies from the original 96-well microtiter plate werere-arrayed into “functionally-corrected” plates (e.g., the cellsidentified as having functional gRNAs were transferred into a 96-wellplate).

Library Re-Amplification, Cloning, and Validation:

Cells identified as having functional gRNAs were pooled and DNA wasextracted and isolated. Serial dilutions were made of the isolated DNA,and amplification was conducted with primers designed to amplify theediting cassettes. PCR was performed under the following conditions: 95°C. for 1 minute; 18 rounds of 95° C. for 30 seconds/60° C. for 30seconds/72° C. for 2.5 minutes; with a final hold at 72° C. for 5minutes. Amplicons were checked on a 1% agarose gel and pools with thecleanest output(s) were identified. The amplified cassettes weremini-prepped and eluted into 50 μL ddH₂O. Next, an isothermal nucleicacid assembly reaction containing 150 ng backbone DNA with 100 ngcassette inserts was performed. An equal volume 2× Master Mix was addedto the backbone and insert, and the reaction was incubated for 45 min @50° C., then dialyzed for 30 min in sitting droplet with 0.25 μm filterdisc. 100 ng of the isothermal nucleic acid assembly reaction wastransformed by electroporation into competent EC1 cells containing theengine vector as described above. Following transformation, the cellswere allowed to recover for 3 hours in SOB medium. A 10-fold dilutionseries of recovered cells (in H₂O) was spot plated and the resulting CFUcounts/dilution ratios were used to calculate transformation efficiency.100 μL of an appropriate dilution of cells were plated on LB medium+25μg/mL chlor and grown at 30° C. overnight. Editing wasassessed/validated by, e.g., sequencing.

Example 6: Enrichment of Editing Cells by Growth Lag Identification

Transformation:

100 ng of the editing vector cloned library or isothermal nucleic acidassembly reaction was transformed by electroporation into 100 μLcompetent EC1 cells containing the engine vector. The electroporator wasset to 2400 V in 2 mm cuvette. Following transformation, the cells wereallowed to recover for 3 hours in SOB medium. A 10-fold dilution seriesof recovered cells (in H₂O) was spot plated and the resulting CFUcounts/dilution ratios were used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB agar mediumcontaining 25 μg/mL chlor and +1% arabinose and grown at 30° C. for 6-8hours. Alternatively, the cells may be grown in liquid culture in LBmedium+25 μg/mL chlor at 30° C. to saturation and diluted to theappropriate concentration before plating on LB agar medium containing 25μg/mL chlor+1% arabinose and grown at 30° C. for 6-8 hours. Followingthe 6-8 hour growth, the temperature of the plate was adjusted to 42° C.and the plates were incubated for two hours. The temperature was thenadjusted back to 30° C. and the cells were allowed to recover overnight.

Edited Cell Identification:

Small-size colonies were identified. The small colony-size phenotypeindicates cell viability was compromised during the induced-editingprocedure. Efficient recovery of edited cells from the initial pool wasaccomplished by identifying and picking small colonies and arrayingcells from these small colonies onto a 96-well plate to create a libraryof edited cells, or the cells from the colonies were pooled generatinghighly-edited cell populations for recursive editing. Editing wasassessed/validated by sequencing. It was found that 85% of the smallcolonies were edited cells.

Example 7: Assessing Loading and Normalization of E. Coli in a SolidWall Device

Electrocompetent E. coli cells were transformed with a cloned library,an isothermal assembled library, or a process control sgRNA plasmid(escapee surrogate) as described in Examples 2-5 above. The E. colistrain carried the appropriate endonuclease and lambda red componentsand editing induction system (e.g., on an engine plasmid or integratedinto the bacterial genome or a combination). Transformations routinelyused 150 ng of plasmid DNA (or isothermal nucleic acid assemblyreactions) with 150 ng of pL sgRNA backbone DNA. Followingelectroporation, the cells were allowed to recover in 3 mL SOB andincubated at 30° C. with shaking for 3 hours. In parallel withprocessing samples through the solid wall device, samples were alsoprocessed with the solid plating protocol (see Example 10 above), so asto compare “normalization” in the sold wall device with the standardbenchtop process. Immediately before the cells were introduced to thepermeable-bottom solid wall device, the 0.2 μm filter forming the bottomof the microwells was treated with a 0.1% TWEEN solution to effectproper spreading/distribution of the cells into the microwells of thesolid wall device. The filters were placed into a Swinnex Filter Holder(47 mm, Millipore®, SX0004700) and 3 mL of a solution with 0.85% NaCl,and 0.1% TWEEN was pulled through the solid wall device and filterthrough using a vacuum. Different TWEEN concentrations were evaluated,and it was determined that for a 47 mm diameter solid wall device with a0.2 μM filter forming the bottom of the microwells, a pre-treatment ofthe solid wall device+filter with 0.1% TWEEN was preferred (data notshown).

After the 3-hour recovery in SOB, the transformed cells were diluted anda 3 mL volume of the diluted cells was processed through theTWEEN-treated solid wall device and filter, again using a vacuum. Thenumber of successfully transformed cells was expected to beapproximately 1.0 E+06 to 1.0 E+08, with the goal of loadingapproximately 10,000 transformed cells into the current 47 mmpermeable-bottom solid wall device (having ˜30,000 wells). Serialdilutions of 10⁻¹, 10⁻², and 10⁻³ were prepared, then 100 μL volumes ofeach of these dilutions were combined with 3 mL 0.85% NaCl, and thesamples were loaded onto solid wall devices. Each permeable-bottom solidwall device was then removed from the Swinnex filter holder andtransferred to an LB agar plate containing carbenicillin (100 μg/mL),chloramphenicol (25 μg/mL) and arabinose (1% final concentration). Thesolid wall devices were placed metal side “up,” so that thepermeable-bottom membrane was touching the surface of the agar such thatthe nutrients from the plate could travel up through the filter “bottom”of the wells. The solid wall devices on the LB agar plates wereincubated for 9 hours at 30° C., at 42° C. for 2 hours, then returned toincubation at 30° C., for 12-16 hour, and, in another experiment for36-40 hours.

At the end of the incubation the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (data notshown). To retrieve cells from the permeable-bottom solid wall device,the filter was transferred to a labeled sterile 100 mm petri dish whichcontained 15 mL of sterile 0.85% NaCl, then the petri dish was placed ina shaking incubator set to 30° C./80 RPM to gently remove the cells fromthe filter and resuspend the cells in the 0.85% NaCl. The cells wereallowed cells to shake for 15 minutes, then were transferred to asterile tube, e.g., a 50 mL conical centrifuge tube. The OD600 of thecell suspension was measured and at this point, the cells can beprocessed in different ways depending on the purpose of the study. Forexample, if the plasmids or libraries are designed to target a sugarmetabolism pathway gene such as galK, then the resuspended cells can bespread onto MacConkey agar plates containing galactose (1% finalconcentration) and the appropriate antibiotics. On this differentialmedium, colonies that are the result of successfully-edited cells areexpected to be phenotypically white in color, whereas unedited coloniesare red in color. This red/white phenotype can then be used to assessthe percentage of edited cells and the extent of normalization of editedand unedited cells. The results of one experiment are shown below inTable 6. In all replicates, the transformed cells were allowed to growin the solid wall devices for 9 hours at 30° C., 2 hours at 42° C., andovernight at 30° C.

TABLE 6 Dilution Red White Tween? counted colonies colonies % edit Notween 10⁻⁴ 72 5   6% No tween 10⁻⁴ 89 3   3% No tween 10⁻³ 64 5   7%Pre-treatment tween 10⁻⁴ 71 5   7% Pre-treatment tween 10⁻³ 443 29   6%Pre-treatment tween 10⁻³ 149 12   7% Pre-treatment tween 10⁻³ 83 21  20% Pre-treatment tween 10⁻² 318 112   26% Pre-treatment tween + tweenin 10⁻³ 163 25   13% cell loading buffer Pre-treatment tween + tween in10⁻⁴ 132 10 7% cell loading buffer Pre-treatment tween + tween in 10⁻⁴31 9   23% cell loading buffer Pre-treatment tween + tween in 10⁻³ 14718 10.9% cell loading buffer Pre-treatment tween + tween in 10⁻² 720 150  17% cell loading buffer Pre-treatment tween + tween in 10⁻³ 55 15  21% cell loading buffer

FIG. 16 is a graph showing the extent of normalization of cells (%edited cells) for different dilutions of transformed cells, and notreatment with TWEEN vs. pre-treatment with TWEEN vs. pre-treatment withTWEEN+TWEEN in the buffer when loading the cells into the microwells ofthe solid wall device. A standard plating protocol (SPP) was conductedin parallel with the solid wall isolation experiments as a benchmark(first bar on the left in the graph). Note that the percentage of editsfor the standard plating protocol was approximately 27.5%, and thepercentage of edits for two replicates of the 10⁻³ dilution of cellswith pre-treatment with TWEEN was approximately 20% and 26%,respectively.

Example 8: Isolation of Yeast Colonies in a Solid Wall Device

Electrocompetent Saccharomyces cerevisiae cells were prepared asfollows: The afternoon before transformation was to occur, 10 mL of YPADwas inoculated with the selected Saccharomyces cerevisiae strain. Theculture was shaken at 250 RPM and 30° C. overnight. The next day, 100 mLof YPAD was added to a 250-mL baffled flask and inoculated with theovernight culture (around 2 mL of overnight culture) until the OD600reading reached 0.3+/−0.05. The culture was placed in the 30° C.incubator shaking at 250 RPM and allowed to grow for 4-5 hours, with theOD checked every hour. When the culture reached an OD600 ofapproximately 1.5, 50 mL volumes were poured into two 50-mL conicalvials, then centrifuged at 4300 RPM for 2 minutes at room temperature.The supernatant was removed from all 50 ml conical tubes, while avoidingdisturbing the cell pellet. 50 mL of a Lithium Acetate/Dithiothreitolsolution was added to each conical tube and the pellet was gentlyresuspended. Both suspensions were transferred to a 250 mL flask andplaced in the shaker; then shaken at 30° C. and 200 RPM for 30 minutes.After incubation was complete, the suspension was transferred to two50-mL conical vials. The suspensions then were centrifuged at 4300 RPMfor 3 minutes, then the supernatant was discarded.

Following the Lithium Acetate/Dithiothreitol treatment step, coldliquids were used and the cells were kept on ice until electroporation.50 mL of 1 M sorbitol was added and the pellet was resuspended,centrifuged at 4300 RPM, 3 minutes, 4° C., after which the supernatantwas discarded. The 1M sorbitol wash was repeated twice for a total ofthree washes. 50 μL of 1 M sorbitol was added to one pellet, cells wereresuspended, then transferred to the other tube to suspend the secondpellet. The volume of the cell suspension was measured and brought to 1mL with cold 1 M sorbitol. At this point the cells were electrocompetentand could be transformed with a cloned library, an isothermal assembledlibrary, or process control sgRNA plasmids. In brief, a required numberof 2-mm gap electroporation cuvettes were prepared by labeling thecuvettes and then chilling on ice. The appropriate plasmid—or DNAmixture—was added to each corresponding cuvette and placed back on ice.100 μL of electrocompetent cells was transferred to each labelledcuvette, and each sample was electroporated using appropriateelectroporator conditions. 900 μL of room temperature YPAD Sorbitolmedia was then added to each cuvette. The cell suspension wastransferred to a 14 mL culture tube and then shaken at 30° C., 250 RPMfor 3 hours. After a 3 hour recovery, 9 mL of YPAD containing theappropriate antibiotic, e.g., geneticin or Hygromycin B, was added.

At this point the transformed cells were processed in parallel in thesolid wall device and the standard plating protocol (see Example 10above), so as to compare “normalization” in the solid wall device withthe standard benchtop process. Immediately before cells the cells wereintroduced to the permeable-bottom solid wall device, the 0.45 μm filterforming the bottom of the microwells (note that a larger-pore filter isused for yeast) was treated with a 0.1% TWEEN solution to effect properspreading/distribution of the cells into the microwells of the solidwall device. The filters were placed into a Swinnex Filter Holder (47mm, Millipore®, SX0004700) and 3 mL of a solution with 0.85% NaCl and0.1% TWEEN was pulled through the solid wall device and filter throughusing a vacuum. Different TWEEN concentrations were evaluated, and itwas determined that for a 47 mm diameter solid wall device with a 0.45μm filter forming the bottom of the microwells, a pre-treatment of thesolid wall device+filter with 0.1% TWEEN was preferred (data not shown).

After the 3-hour recovery in SOB, the transformed cells were diluted anda 3 mL volume of the diluted cells was processed through theTWEEN-treated solid wall device and filter, again using a vacuum. Thenumber of successfully transformed cells was expected to beapproximately 1.0 E+06 to 1.0 E+08, with the goal of loadingapproximately 10,000 transformed cells into the current 47 mmpermeable-bottom solid wall device (having ˜30,000 wells). Serialdilutions of 10⁻¹, 10⁻², and 10⁻³ were prepared, then 100 μL volumes ofeach of these dilutions were combined with 3 mL 0.85% NaCl, and thesamples were loaded onto solid wall devices. Each permeable-bottom solidwall device was then removed from the Swinnex filter holder andtransferred to an LB agar plate containing carbenicillin (100 μg/mL),chloramphenicol (25 μg/mL) and arabinose (1% final concentration). Thesolid wall devices were placed metal side “up,” so that thepermeable-bottom membrane was touching the surface of the agar such thatthe nutrients from the plate could travel up through the filter “bottom”of the wells. The solid wall devices on the YPD agar plates wereincubated for 2-3 days at 30° C.

At the end of the incubation, the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (see FIGS.17A and 17B). To retrieve cells from the permeable-bottom solid walldevice, the filter was transferred to a labeled sterile 100 mm petridish which contained 15 mL of sterile 0.85% NaCl, then the petri dishwas placed in a shaking incubator set to 30° C./80 RPM to gently removethe cells from the filter and resuspend the cells in the 0.85% NaCl. Thecells were allowed to shake for 15 minutes, then were transferred to asterile tube, e.g., a 50 mL conical centrifuge tube. The OD600 of thecell suspension was measured; at this point the cells can be processedin different ways depending on the purpose of the study. For example, ifan ADE2 stop codon mutagenesis library is used, successfully-editedcells should result in colonies with a red color phenotype when theresuspended cells are spread onto YPD agar plates and allowed to growfor 4-7 days. This phenotypic difference allows for a quantification ofpercentage of edited cells and the extent of normalization of edited andunedited cells.

Example 9: Protocol Flow from mTFF to FTEP to SWIIN

mTFF Module, E. coli Workflow:

Approximately 20 ml of E. coli was transferred from a rotating growthvial (RGV) in a cell growth module. Specially, the E. coli was EC83, anE. coli MG1655 strain comprising an engine vector coding for the λRedrecombineering system and a MAD7 coding sequence (see, e.g., FIG. 11A).In the RGV, the EC83 was grown in LB growth medium to an OD600 ˜0.6 andhaving a conductivity of ˜16,500 μS/cm. In the mTFF the cells werewashed with a low-conductivity solution (10% glycerol) and concentratedin a small volume (approximately 0.80 ml) in the same low-conductivitysolution. Both the input and output cell counts were determined byplating on solid media. The cell input was approximately 3.1 E+09 andthe cell output was approximately 2.3 E+09. The output of the mTFF wasused as input for the flow-through electroporation (FTEP) module.

FTEP Module, E. coli Workflow:

Approximately 500 μl of the concentrated EC83 cells in 10%glycerol—e.g., an aliquot of the output of the mTFF—was combined with100 μl of the assembled editing vector (see, e.g., FIG. 11B) and a TWEENsolution (see Example 7 above). The cells and DNA were mixed and passedthrough the FTEP at a very high field strength in a high resistancesolution. After transformation in the FTEP, the cells were transferredback to the cell growth module into a fresh RGV containing 3 ml SOB withchloramphenicol. The cells were incubated at 30° C. for 1 hour forrecovery. The input CFU was approximately 2.3 E+09 and the output CFUwas approximately 9.8 E+08 survival and 8.5 E+05 uptake. SWIIN module,E. coli workflow: Subsequent to the 1 hour recovery in the cell growthmodule, approximately 0.35 mL of the cells were combined withapproximately 9.5 mL of a PBS/TWEEN solution and the cells were loadedonto the SWIIN. Once cells were loaded and growth medium (was LB with 1%arabinose, 25 ug/ml chloramphenicol and 100 μg/ml carbenicillin) wasadded to the permeate chamber of the SWIIN, the SWIIN was placed in aprogrammable incubator for the induction and editing stages. The scriptfor the SWIIN protocol was set for a 9 hour incubation at 30° C., a 2.5hour incubation at 42° C., then 9 additional hours at 30° C. The CFUinput into the SWIIN was approximately 7.5 E+04 and the CFU output wasapproximately 6.5 E+06 in a 7.0 ml volume. To recover the cells from theSWIIN, the retentate channel was flooded to dislodge the cells withpositive pressure applied to the permeate reservoirs, thus pushing fluidfrom the permeate reservoirs into the retentate reservoirs. Followingthis “push,” all fluid was swept to one of the retentate reservoirs byapplying a vacuum to the other retentate reservoir. Finally, the cellsolution from retentate reservoir into which the fluid was swept wastransferred into a vial, and all liquid was aspirated out of bothretentate reservoirs. (Also see Example 10, below.)

Amplicon and Singleplex Sequencing:

Cells were recovered from the SWIIN with a PBS solution, and 100 μl ofthe undiluted cell suspension was spread on an LB chlor/carb agar plateand incubated overnight at 30° C. This plate was then “scraped” and usedas the amplicon DNA input. Dilutions were also prepared from thePBS/cell suspension and then plated on an LB chlor/carb agar plate andincubated overnight at 30° C.; isolated colonies were selected and usedfor singleplex sequencing.

In addition to loading, growing and editing cells on the SWIIN module,curing can be performed on the SWIIN module as well. (See, e.g., Example12, below.)

Example 10: Loading and Performing Editing on a SWIIN

FIGS. 4AA 4DD are simplified overviews of various parameters for loadingcells onto a SWIIN module, performing editing, and removing orrecovering the edited cells from the SWIIN module. The steps of FIGS.4AA-4DD correspond to the steps listed in Tables 3-5, with the exceptionthat step 1 (loading the SWIIN module into the automated multi-modulecell processing instrument) and step 32 (unloading the SWIIN from theautomated multi-module cell processing instrument) are not representedon FIGS. 4AA-4DD. FIG. 4AA begins with step 2, where 10 mL of PBS/0.01%Tween80 was transferred from a reagent cartridge to permeate reservoir1. At this initial step, retentate reservoir 1, retentate reservoir 2,and permeate reservoir 1 were under positive pressure, flow sensor 1detected a high flow rate and flow sensor 2 detected a low flow rate. Atstep 3, additional PBS/0.01% Tween80 was loaded into the permeatechannel and a bubble flush was performed. At step 4, more PBS/0.01%Tween80 was loaded into the permeate channel to fill the permeatechannel. Step 4 ended with a flow meter trigger, and flow sensor 1returned to baseline once the permeate channel was filled with liquid.

At step 5, a vacuum was applied at retentate reservoirs 1 and 2, and theretentate channel was flooded. Once the retentate channel was flooded(and there was minimal fluid remaining in the permeate reservoirs), thenegative pressure (vacuum) was removed. At step 6, negative pressure wasapplied to retentate reservoir 2, thereby sweeping all liquid toretentate reservoir 2. At step 7, the liquid in retentate reservoirs 1and 2 was removed by, e.g., an air displacement pipette. At step 8, 9.5mL of PBS/0.01% Tween80 was transferred from the reagent cartridge toretentate reservoir 1, and at step 9, 0.5 mL of transformed cells weretransferred from the transformation module (the flow-throughelectroporation device) to retentate reservoir 1. At step 10, thecell-containing liquid was pipetted up and down in retentate reservoir1, and at step 11, the cell-containing liquid was pulled from retentatereservoir 1 into the retentate channel leaving minimal fluid inretentate reservoirs 1 and 2. Step 11 is sensitive to timing and wasthus controlled via air displacement pipette liquid level detection inretentate reservoir 1.

FIG. 4BB begins with step 12, in which the fluid in the serpentinechannel in the retentate member was pulled through the membrane orfilter member on low vacuum; that is, negative pressure was applied toboth permeate reservoirs 1 and 2. At step 13, the fluid in theserpentine channel in the retentate member was pulled through the filtermember on high vacuum, with fluid pulled into the serpentine channel inthe permeate member and then into permeate reservoirs 1 and 2. At step14, all fluid was swept to permeate reservoir 1 by applying positivepressure to retentate reservoirs 1 and 2 and permeate reservoir 2.Remaining liquid was aspirated out of permeate reservoirs 1 and 2 atstep 5, with the air displacement pipette accessing permeate reservoir1. At step 16, 10 mL medium was transferred from the reagent cartridgeto permeate reservoir 1, and at step 17, the medium was transferred frompermeate reservoir 1 into the permeate channel. Step 17 is sensitive totiming and thus was controlled via liquid level detection via a airdisplacement pipette in retentate reservoir 1. At the end of step 17,some amount of liquid resided in permeate reservoir 2, and at step 18,liquid (permeate) was aspirated out of permeate reservoirs 1 and 2.

Steps 19-23 are not represented in FIGS. 4AA-4DD. At step 19, the SWIINmodule was incubated at 30° C. with intermittent airflow and mediumrinses/exchanges, as deemed necessary. In step 20, the temperature ofthe SWIIN module was raised to 42° C., and at step 21 the SWIIN modulewas incubated for 2 hours. At step 22, the temperature of the SWIINmodule was ramped down from 42° C. to 30° C., and the SWIIN was thenincubated at 30° C. for 9 hours. During this incubation the manifoldarms of the SWIIN assembly may be unsealed and resealed to effectairflow. In addition, media rinses or exchanges may be performed.

FIG. 4CC begins with step 24, where medium was pulled out of thepermeate channel into permeate reservoir 2, by applying negativepressure to permeate reservoir 2. During this time, the flow rate forflow sensor 1 and flow sensor 2 are roughly the same at 0, then flowsensor 2 spiked to 0, rebounded, then spiked to 0 again triggering flowsensor 2. Step 24 ended with fluid in permeate reservoir 2. At step 25,the liquid was aspirated out of permeate reservoir 2 by applying avacuum to permeate reservoir 2, and at step 26, 10 mL of mediumcontaining 10% glycerol was transferred from the reagent cartridge topermeate reservoir 1. At step 27 the medium/10% glycerol was pulled frompermeate reservoir 1 into the permeate channel, and at the end of thisstep, a minimal amount of fluid remains in permeate reservoir 2. At step28 (see FIG. 4DD), the retentate channel was flooded (filled) todislodge the cells with positive pressure applied to permeate reservoirs1 and 2, thus pushing fluid from the permeate reservoirs into theretentate reservoirs. Next at step 29, all fluid was swept to retentatereservoir 2 by applying a vacuum to retentate reservoir 2. Step 29 iscontrolled by the trigger of flow sensor 1. Step 30 involved aspiratingthe cell solution from retentate reservoir 2 into a vial, and step 31involved aspirating all liquid out of both retentate reservoirs. Thefinal step, step 32, is not represented on FIG. 4DD, but involvedremoving the SWIIN from the automated multi-module cell processinginstrument.

Example 11: Isolation, Growth and Editing of E. coli in 200K SWIIN

Singleplex automated genomic editing using MAD7 nuclease, a library with94 different edits in a single gene (yagP) and employing a 200K SWIINmodule (i.e., a SWIIN module with approximately 200K wells) such asthose exemplified in FIGS. 4F-4R was successfully performed. The enginevector used was substantially similar to that depicted in FIG. 11A (withMAD7 under the control of the pL inducible promoter), and the editingvector used was substantially similar to that depicted in FIG.11B—including the editing cassette being under the control of the pLinducible promoter, and the λ Red recombineering system under control ofthe pBAD inducible promoter pBAD—with the exception that the editingcassette comprises the 94 yagP gene edits (donor DNAs) and theappropriate corresponding gRNAs. Two SWIIN workflows were compared, andfurther were benchmarked against the standard plating protocol (seeExample 7). The SWIIN protocols different from one another that in oneset of replicates LB medium containing arabinose was used to distributethe cells in the SWIIN (arabinose was used to induce the λ Redrecombineering system (which allows for repair of double-strand breaksin E. coli that are created during editing), and in the other set ofreplicates SOB medium without arabinose was used to distribute the cellsin the SWIIN and for initial growth, with medium exchange performed toreplace the SOB medium without arabinose with SOB medium with arabinose.Approximately 70K cells were loaded into the 200K SWIIN.

In all protocols (standard plating, LB-SWIIN, and SOB-SWIIN), the cellswere allowed to grow at 30° C. for 9 hours and editing was induced byraising the temperature to 42° C. for 2.5 hours, then the temperaturewas returned to 30° C. and the cells were grown overnight. The resultsof this experiment are shown in FIG. 18 and in Table 7 below. Note thatsimilar editing performance was observed with the four replicates of thetwo SWIIN workflows, indicating that the performance of SWIIN platingwith and without arabinose in the initial medium is similar. Editingpercentage in the standard plating protocol was approximately 77%, inbulk liquid was approximately 67%, and for the SWIIN replicates rangedfrom approximately 63% to 71%. Note that the percentage of unique editcassettes divided by the total number of edit cassettes was similar foreach protocol.

TABLE 7 SWIIN SWIIN SWIIN SWIIN SOB then SOB then Standard LB/Ara LB/AraSOB/Ara SOB/Ara Plating Rep. A Rep. B Rep. A Rep. B 40006 editcalls/identified wells 0.777 0.633 0.719 0.663 0.695 Unique editcassettes/total 0.49 0.49 0.43 0.50 0.51 edit cassettes

Example 12: Curing on the SWIIN

Experiments were carried out to determine whether curing could beperformed on the SWIIN. “Curing” is a process in which one or moreediting vectors used in a prior round of editing is eliminated from theedited cells, or, as in this Example, a process in which the enginevector is removed from edited cells at the end of the editing process.Curing can be accomplished by “active” curing, which refers to cleavinga vector at a curing target sequence. In active curing, in addition to acuring target sequence, there is also provided a genetic element (forexample, a curing gRNA) on the vector to be cured or on another vectorthat targets the curing target sequence with the result of rendering thevector to be cured nonfunctional by, e.g., double-stranded that is notrepaired. Curing can also be accomplished by “passive” curing whichinvolves diluting the vector(s) to be cured in the cell population viacell growth; that is, the more growth cycles the cells go through, thefewer daughter cells will retain the editing or engine vector(s)).Passive curing can also be accomplished by, e.g., utilizing aheat-sensitive origin of replication on the vector to be cured, whichessentially prevents the vector from replicating.

In this curing protocol, the engine vector was “passively” cured onceediting had taken place. Curing of the engine vector after one or moreediting steps is desired to rid the cell population of the codingsequence for the nuclease. In the curing protocol used in this Example12, E. coli cells were transformed with an engine vector very similar tothat described in relation to FIG. 11A in Example 1 above. The enginevector comprised the MAD7 nuclease coding sequence under the control ofthe inducible pL promoter (induced by an increase in temperature; e.g.,see FIG. 1C and the description thereof supra), the Red recombineeringsystem under the control of the pBAD promoter (induced by the additionof arabinose to the growth medium), and a selective marker comprisingthe coding sequence for chloramphenicol resistance. The “curing” enginevector used in this Example 12, however, also comprised a heat-sensitiveorigin of replication, which when the temperature is raised to 42° C.fails to bind the protein needed to replicate the engine vector.Preventing replication of the engine vector combined with active celldivision thereby effectively dilutes the engine vector from the cellpopulation. The editing vector used in this Example 12 was an editingvector similar to that described in relation to FIG. 11B in Example 1above, where an edit changed coding for an amino acid residue in theXylA gene to a stop coding, allowing for a phenotypic read out to showediting.

Protocols for preparing E. coli cells for editing are described inExample 9 above; a step-wise protocol for loading, performing editing,and unloading a SWIIN is described in Example 10 above; and protocolsfor isolation, growth and editing on the SWIIN is described in Example11 above. Four different curing protocols were performed after cellediting on the SWIIN. Editing was performed with chloramphenicol in thegrowth medium to select for the engine vector. The first curing protocolcomprised recovering cells from the SWIIN and growing the recoveredcells in bulk liquid (without chloramphenicol) at 42° C. for six hours.The second curing protocol comprised recovering cells from the SWIIN andloading and growing the recovered cells on a fresh SWIIN (withoutchloramphenicol) at 42° C. for six hours. The third curing protocolcomprised recovering a fraction of the cells from the SWIIN to make roomfor additional cell growth, performing medium exchange on the SWIIN formedium without chloramphenicol, then continuing to grow the cells on theSWIIN at 42° C. for six hours. The fourth curing protocol like the thirdcuring protocol did not comprise a cell recovery step; instead, thecells remained on the SWIIN, medium exchange was performed, and thecells were grown at 42° C. for six hours. (Note that the data reportedin FIG. 13 described above demonstrates that cell viability is notimpacted by raising the temperature to 42° C.)

The curing efficiency (%) for each of these curing protocols is shown inFIG. 19. Note all protocols provided a curing efficiency of greater than85%, where the first and second protocols provided a curing efficiencyof 100%, the third protocol provided a curing efficiency ofapproximately 97%, and the fourth protocol provided a curing efficiencyof approximately 85.5%.

In addition to passive curing on the SWIIN, active curing may beperformed as well, where, e.g., editing vectors are cleared betweenrounds of editing. In active curing, a sequence on the vector to becured (e.g., the editing vector between rounds of editing) comprises acuring target sequence that is subjected to a double-stranded cut by,e.g., a curing gRNA. See, e.g., U.S. Ser. No. 62/857,967, filed 6 Jun.2019.

Example 13: Identification of Edits Proximal to Non-Canonical PAMs

As described above, because the present methods and modules allow editedcells to be detected in a background of unedited cells, cellular targetsites located near non-canonical PAMs can be detected. The presentexperiments were performed with MAD7, a nucleic acid-guided nucleasethat prefers the T-rich PAMs TTTV and CTTV. FIG. 20A is a depletion plotshowing that there is reduced activity at TCTV PAMS in E coli, and apreference for TCTG over TCTA or TCTC. FIG. 20B is a bar graph showingTCTV PAMs lead to low efficiency editing from randomly-picked coloniesfollowing standard plating-based editing using a library targeting thegalK locus in E. coli. The measurements correspond to the fraction ofcolonies with white phenotype on Mackonkey agar supplemented withgalactose. Cherry-picking small colonies yielded an approximate 5-6×gain in efficiency in identifying cells with edits located at TCTV PAMs.Sanger sequencing of the genomic amplicons from these edited clonesconfirmed TCTV PAM targeting, with many in the TCTG target set (data notshown). Table 8 below shows the PAM/Spacer sequences for which editingwas observed in this experiment.

TABLE 8 Distance Target Spacer from PAM Cassette Description PAM SpacerSequence GC Content to cut galK_C46_TGC- TCTG CCCTGCGCGATTGATTATCAA 0.4813 TAA_TCTG-GTTA_145_7 SEQ ID No. 1 galK_A47_GCG- TCTGCCCTGCGCGATTGATTATCAA 0.48 10 TAA_TCTG-GTTA_145_10 SEQ ID No. 2galK_I48_ATT- TCTG CCCTGCGCGATTGATTATCAA 0.48  7 TAA_TCTG-GTTA_155_13SEQ ID No. 3 galK_R97_CGT- TCTG CAACTGCGTAACAACAGCTTC 0.48 37TAA_TCTG-GTTA_145_17 SEQ ID No. 4 galK_D150_GAC- TCTGCCGCTCCACGGCGCACAAATC 0.67 10 TAA_TCTG-GTTA_145_10 SEQ ID No. 5galK_A161_GCA- TCTG CCGCTGGACGGCGCACAAATC 0.57 16 TAA_TCTG-GTTA_145_7SEQ ID No. 6

Example 14: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument such asthat shown in FIGS. 5A-5D. See U.S. Pat. No. 9,982,279, issued 29 May2018 and Ser. No. 10/240,167, issued 9 Apr. 2019.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via isothermal nucleic acid assembly into an “editing vector”in an isothermal nucleic acid assembly module included in the automatedinstrument. lacZ_F172 functionally knocks out the lacZ gene.“lacZ_F172*” indicates that the edit happens at the 172nd residue in thelacZ amino acid sequence. Following assembly, the product was de-saltedin the isothermal nucleic acid assembly module using AMPure beads,washed with 80% ethanol, and eluted in buffer. The assembled editingvector and recombineering-ready, electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) and allowed to recover inSOC medium containing chloramphenicol. Carbenicillin was added to themedium after 1 hour, and the cells were allowed to recover for another 2hours. After recovery, the cells were held at 4° C. until recovered bythe user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0 E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example 15: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via isothermal nucleic acidassembly into an “editing vector” in an isothermal nucleic acid assemblymodule included in the automated system. Similar to the lacZ_F172 edit,the lacZ_V10 edit functionally knocks out the lacZ gene. “lacZ_V10”indicates that the edit happens at amino acid position 10 in the lacZamino acid sequence. Following assembly, the product was de-salted inthe isothermal nucleic acid assembly module using AMPure beads, washedwith 80% ethanol, and eluted in buffer. The first assembled editingvector and the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid residue, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers are performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

TABLE 1 SWIIN Design 1 Valve Status and Pressure Manifold/ SV1- SV2-SV3- SV4- SV5- SV6- SV7- SV8- SV9- SV10- Description of step Step armpump PR1 PR2 RR1 RR2 PR3 PR4 PR5 RR4 PUMP “P” PUMP “V” psi Load SWIIN 1open 0 0 0 0 0 0 0 0 0 0 0 0 module on instrument Load 7.5 ml of 2 open0 0 0 0 0 0 0 0 0 0 0 0 cell suspension into RR1 and RR3 Fill retentate3 closed 1 0 0 1 0 0 0 1 0 0 0 0.5 layer with cell suspension Let cellssettle to 4 closed 0 0 0 0 0 0 0 0 0 0 0 0 the vicinity of theperforated sheet and into microwells Drive medium in 5 closed 1 1 1 0 01 1 0 0 1 1 −5 the retentate layer across membrane Open manifold 6 open0 0 0 0 0 0 0 0 0 0 0 0 Remove excess 7 open 0 0 0 0 0 0 0 0 0 0 0 0medium from PRs with pipettor Load 7.5 ml 8 open 0 0 0 0 0 0 0 0 0 0 0 0growth medium into PR1 and PR3 Close manifold 9 closed 0 0 0 0 0 0 0 0 00 0 0 Flush permeate 10 closed 0 0 0 0 0 0 0 0 0 0 0 0 layer with growthmedium from PR1 to PR2 and PR3 Open manifold 11 open 0 0 0 0 0 0 0 0 0 00 0 Remove excess 12 open 0 0 0 0 0 0 0 0 0 0 0 0.5 medium from PRs withpipettor Load 7.5 ml 13 open 0 0 0 0 0 0 0 0 0 0 0 0 growth medium intoPR1 and PR3 Close manifold 14 closed 0 0 0 0 0 0 1 0 0 1 1 −5 Flushpermeate 15 closed 0 0 0 0 0 0 0 0 0 0 0 0 layer with growth medium fromPR1 to PR2 and PR3 to PR4 via gravity Open manifold 16 open 0 0 0 0 0 00 0 0 0 0 0 Remove excess 17 open 0 0 0 0 0 0 0 0 0 0 0 0 medium fromPRs with pipettor Close manifold 18 closed 0 0 0 0 0 0 0 0 0 0 0 0Incubate at 30° C. 19 closed 1 0 0 1 0 0 0 1 0 0 0 0.25 for 4.5 hoursOpen manifold 20 open 0 0 0 0 0 0 0 0 0 0 0 0 Load 7.5 ml 21 open 0 0 00 0 0 0 0 0 0 0 0 growth medium into PR1 and PR3 Close manifold 22closed 0 0 0 0 0 0 0 0 0 0 0 0 Incubate at 30° C. 23 closed 1 0 0 1 0 00 1 0 0 0 0.25 for 4.5 hours Incubate at 42° C. 24 closed 1 0 0 1 0 0 01 0 0 0 0.25 for 2 hours Incubate at 30° C. 25 closed 1 0 0 1 0 0 0 1 00 0 0.25 for 9 hours Aspirate cells 26 closed 1 0 0 1 0 0 1 0 0 1 1 −5into RR1 and RR2 Open manifold 27 open 1 0 0 0 0 0 0 0 0 0 0 0 Recovercells 28 open 1 0 0 0 0 0 0 0 0 0 0 0 from RR1 and RR3 with pipettorRemove 29 open 1 0 0 0 0 0 0 0 0 0 0 0 remaining medium from PR1 and PR2and PR3 and PR4

TABLE 2 SWIIN Design 1: Reservoir Volumes RR1 and RR3 RR2 and RR4 PR1and PR3 PR2 and PR4 Temperature Description of step Step Initial/FinalInitial/Final Initial/Final Initial/Final (° C.) Load SWIIN 1 0/0 0/00/0 0/0 25 module on instrument Load 7.5 ml of 2   0/7.5 TBD/TBD 0/0 0/025 cell suspension into RR1 and RR3 Fill retentate 3 7.5/0   TBD/TBD 0/00/0 25 layer with cell suspension Let cells settle to 4 0/0 TBD/TBD 0/00/0 4 the vicinity of the perforated sheet and into microwells Drivemedium in 5 0/0 TBD/TBD 0/1 0/1 30 the retentate layer across membraneOpen manifold 6 0/0 TBD/TBD 1/1 1/1 30 Remove excess 7 0/0 TBD/TBD 1/01/0 30 medium from PRs with pipettor Load 7.5 ml 8 0/0 TBD/TBD   0/7.50/0 30 growth medium into PR1 and PR3 Close manifold 9 0/0 TBD/TBD7.5/7.5 0/0 30 Flush permeate 10 0/0 TBD/TBD  7.5/3.75   0/3.75 30 layerwith growth medium from PR1 to PR2 and PR3 Open manifold 11 0/0 TBD/TBD3.75/3.75 3.75/3.75 30 Remove excess 12 0/0 TBD/TBD 3.75/0   3.75/0   30medium from PRs with pipettor Load 7.5 ml 13 0/0 TBD/TBD   0/7.5 0/0 30growth medium into PR1 and PR3 Close manifold 14 0/0 TBD/TBD 7.5/7.5 0/030 Flush permeate 15 0/0 TBD/TBD  7.5/3.75   0/3.75 30 layer with growthmedium from PR1 to PR2 and PR3 to PR4 via gravity Open manifold 16 0/0TBD/TBD 3.75/3.75 3.75/3.75 30 Remove excess 17 0/0 TBD/TBD 3.75/0  3.75/0   30 medium from PRs with pipettor Close manifold 18 0/0 TBD/TBD0/0 0/0 30 Incubate at 30° C. 19 0/0 TBD/TBD 0/0 0/0 30 for 4.5 hoursOpen manifold 20 0/0 TBD/TBD 0/0 0/0 30 Load 7.5 ml 21 0/0 TBD/TBD  0/7.5 0/0 30 growth medium into PR1 and PR3 Close manifold 22 0/0TBD/TBD  7.5/3.75   0/3.75 30 Incubate at 30° C. 23 0/0 TBD/TBD3.75/3.75 3.75/3.75 30 for 4.5 hours Incubate at 42° C. 24 0/0 TBD/TBD3.75/3.75 3.75/3.75 42 for 2 hours Incubate at 30° C. 25 0/0 TBD/TBD3.75/3.75 3.75/3.75 30 for 9 hours Aspirate cells 26 0/5 TBD/TBD3.75/1.25 3.75/1.25 RT into RR1 and RR2 Open manifold 27 5/5 TBD/TBD1.25/1.25 1.25/1.25 RT Recover cells 28 5/0 TBD/TBD 1.25/1.25 1.25/1.25RT from RR1 and RR3 with pipettor Remove 29 0/0 TBD/TBD 1.25/0  1.25/0   RT remaining medium from PR1 and PR2 and PR3 and PR4

TABLE 3 SWIIN Design 2 Valve Status and Prop Valve PSI Prop PropManifold Manifold SV- SV- SV1 SV2 SV3 SV4 Valve 1 Valve 2 Description ofstep Step ARM 1 ARM 2 Pump POS NEG (RR1) (PR1) (PR2) (RR2) (psi) (psi)Load SWIIN 1 open open 0 0 0 0 0 0 0 0 0 cartridge on the instrumentTransfer 10 mL PBS- 2 closed 0 0 0 0 0 0 0 0 0 0 0.01% Tween 80 fromReagent Strip to PR1 Load PBS- 3 closed sealed 1 1 0 0 1 0 0 0.5 0.50.1% Tween 80 into Permeate channel - Bubble Flush Load PBS- 4 closedsealed 1 1 0 1 1 0 1 0.5 0.5 0.1% Tween 80 into Permeate channel - FillChannel Flood Retentate - 5 sealed sealed 1 0 1 1 0 0 1 −0.7 −0.7Symmetrically Apply Vacuum to Retentate Flood Retentate - 6 sealedsealed 1 0 1 0 0 0 1 0 −0.7 Sweep to RR2 Aspirate liquid out of 7 openopen 1 0 0 0 0 0 0 0 0 RR1 & RR2 Transfer 9.5 mL of 8 open closed 1 0 00 0 0 0 0 0 PBS-0.01% Tween 80 from Reagent Strip to RR1 Transfer 0.5 mLcell 9 open closed 1 0 0 0 0 0 0 0 0 Solution from FTEP to RR1 Pipettecell solution 10 open closed 1 0 0 0 0 0 0 0 0 up/down in RR1 Pull cellsolution 11 open sealed 1 0 1 0 0 0 1 0 −0.7 from RR1 into RetentateChannel Pull retentate 12 sealed sealed 1 0 1 0 1 1 0 −0.7 −0.7 throughmembrane (low vac) Pull retentate 13 sealed sealed 1 0 1 0 1 1 0 −1 −1through membrane (high vac) Sweep all fluid to PR1 14 sealed sealed 1 10 1 0 1 1 0.5 0.5 Aspirate liquid out of 15 open open 1 0 0 0 0 0 0 0 0PR1 & PR2 Transfer 10 mL media 16 open closed 1 0 0 0 0 0 0 0 0 fromReagent Strip to PR1 Load media from PR1 17 open closed 1 0 1 0 0 1 0 0−0.7 into Permeate channel Aspirate liquid out of 18 open open 0 0 0 0 00 0 0 0 PR1 & PR2 INCUBATE SWIIN 30 C. 19 closed closed 0 0 0 0 0 0 0 00 #1 - may require intermittent airflow, media rinses Ramp up (30 C. to42 C.) 20 closed closed 0 0 0 0 0 0 0 0 0 INCUBATE SWIIN 42 C.- 21closed closed 0 0 0 0 0 0 0 0 0 may require intermittent airflow, mediarinses Ramp down (42 C. to 30 C.) 22 closed closed 0 0 0 0 0 0 0 0 0INCUBATE SWIIN 30 C. 23 closed closed 0 0 0 0 0 0 0 0 0 #2 - may requireintermittent airflow, media rinses Pull media out of 24 sealed sealed 10 1 0 0 1 0 0 −0.7 Permeate channel into PR2 Aspirate liquid out of 25open open 1 0 0 0 0 0 0 0 0 PR2 Transfer 10 mL 26 open closed 1 0 0 0 00 0 0 0 media + 10% glycerol from Reagent Strip to PR1 Pull media + 10%glycerol 27 open sealed 1 0 1 0 0 1 0 0 −0.7 from PR1 into Permeatechannel Flood Retentate - 28 sealed sealed 1 1 0 0 1 1 0 1 1 DislodgeCells Sweep all fluid to RR2 29 sealed sealed 1 0 1 0 0 0 1 0 −0.7Aspirate 5 mL cell 30 closed open 0 0 0 0 0 0 0 0 0 solution from RR2into final vial Aspirate liquid out of 31 open open 0 0 0 0 0 0 0 0 0 RR& RR2 Unload SWIIN 32 open open 0 0 0 0 0 0 0 0 0

TABLE 4 SWIIN Design 2 Flow Meter Status FM1 (PR2) FM1 (PR2) FM2 (RR2)FM2 (RR2) Valve delay Requires Duration Description of step DetectionThreshold Detection Threshold after spike(s) pLLD (sec) Load SWIIN NA NANA NA NA 0 N/A cartridge on the instrument Transfer 10 mL PBS- NA NA NANA NA 0 as needed 0.01% Tween 80 from Reagent Strip to PR1 Load PBS- NANA NA NA NA 0 0.5 0.1% Tween 80 into Permeate channel - Bubble FlushLoad PBS- NA NA FALL 10 5 0 until FM trigger 0.1% Tween 80 into Permeatechannel - Fill Channel Flood Retentate - NA NA NA NA NA 0 30Symmetrically Apply Vacuum to Retentate Flood Retentate - NA NA NA NA NA0 60 Sweep to RR2 Aspirate liquid out of NA NA NA NA NA 0 determined byRR1 & RR2 ADP Transfer 9.5 mL of NA NA NA NA NA 0 determined byPBS-0.01% Tween 80 ADP from Reagent Strip to RR1 Transfer 0.5 mL cell NANA NA NA NA 0 determined by Solution from FTEP ADP to RR1 Pipette cellsolution NA NA NA NA NA 0 10 up/down in RR1 Pull cell solution NA NA NANA NA 1 until RR1 & RR2 from RR1 into are equal volume Retentate ChannelPull retentate NA NA NA NA NA 0 90 through membrane (low vac) Pullretentate NA NA NA NA NA 0 30 through membrane (high vac) Sweep allfluid to PR1 RISE 50 NA NA 0 0 until FM trigger Aspirate liquid out ofNA NA NA NA NA 0 determined by PR1 & PR2 ADP Transfer 10 mL media NA NANA NA NA 0 determined by from Reagent Strip to ADP PR1 Load media fromPR1 NA NA NA NA NA 1 until PR1 is nearly into Permeate exhausted channelAspirate liquid out of NA NA NA NA NA 0 determined by PR1 & PR2 ADPINCUBATE SWIIN 30 C. NA NA NA NA NA 0 32400 #1 - may requireintermittent airflow, media rinses Ramp up (30 C. to 42 C.) NA NA NA NANA 0 900 INCUBATE SWIIN 42 C.- NA NA NA NA NA 0 7200 may requireintermittent airflow, media rinses Ramp down (42 C. to 30 C.) NA NA NANA NA 0 900 INCUBATE SWIIN 30 C. NA NA NA NA NA 0 32400 #2 - may requireintermittent airflow, media rinses Pull media out of RISE 50 NA NA 0 0until FM trigger Permeate channel into PR2 Aspirate liquid out of NA NANA NA NA 0 determined by PR2 ADP Transfer 10 mL NA NA NA NA NA 0determined by media + 10% glycerol ADP from Reagent Strip to PR1 Pullmedia + 10% glycerol NA NA NA NA NA 1 until PR1 and PR2 from PR1 intoare equal volume Permeate channel Flood Retentate - NA NA NA NA NA 0 30Dislodge Cells Sweep all fluid to RR2 NA NA RISE 50 0 0 until FM triggerAspirate 5 mL cell NA NA NA NA NA 0 determined by solution from RR2 ADPinto final vial Aspirate liquid out of NA NA NA NA NA 0 determined by RR& RR2 ADP Unload SWIIN NA NA NA NA NA 0 N/A

TABLE 5 SWIIN Design 2 Reservoir Volumes RR1 RR1 PR1 PR1 PR2 PR2 RR2 RR2Temperature Description of steps Initial Final Initial 2 Final 2 Initial3 Final 3 Initial 4 Final 4 (° C.) Notes Load SWIIN 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 Continue Ramp cartridge on the Up (RT to 30) instrumentTransfer 10 mL PBS- 0.0 0.0 10.0 10.0 0.0 0.0 0.0 0.0 Continue Ramp0.01% Tween 80 from Up (RT to 30) Reagent Strip to PR1 Load PBS- 0.0 0.010.0 9.8 0.0 0.0 0.0 0.0 Continue Ramp This 0.5 s step consumes 0.1%Tween 80 into Up (RT to 30) very little liquid Permeate channel - BubbleFlush Load PBS- 0.0 0.0 9.8 2.5 0.0 2.5 0.0 0.0 Continue Ramp Requiresdebounce delay 0.1% Tween 80 into Up (RT to 30) for flow sensor to reachPermeate channel - high, trigger threshold Fill Channel untested FloodRetentate - 0.0 2.5 2.5 0.0 2.5 0.0 0.0 2.5 Continue Ramp SymmetricallyApply Up (RT to 30) Vacuum to Retentate Flood Retentate - 2.5 0.0 0.00.0 0.0 0.0 2.5 10.0 Continue Ramp Sweep to RR2 Up (RT to 30) Aspirateliquid out of 0.0 0.0 0.0 0.0 0.0 0.0 10.0 0.0 Continue Ramp RR1 & RR2Up (RT to 30) Transfer 9.5 mL of 0.0 9.5 0.0 0.0 0.0 0.0 0.0 0.0Continue Ramp PBS-0.01% Tween 80 Up (RT to 30) from Reagent Strip to RR1Transfer 0.5 mL cell 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 Continue RampSolution from FTEP Up (RT to 30) to RR1 Pipette cell solution 0.0 10.00.0 0.0 0.0 0.0 0.0 0.0 Continue Ramp up/down in RR1 Up (RT to 30) Pullcell solution 10.0 2.5 0.0 0.0 0.0 0.0 0.0 2.5 Continue Ramp from RR1into Up (RT to 30) Retentate Channel Pull retentate 2.5 0.0 0.0 2.5 0.02.5 2.5 0.0 Continue Ramp through membrane Up (RT to 30) (low vac) Pullretentate 0.0 0.0 2.5 2.5 2.5 2.5 0.0 0.0 Continue Ramp This step isonly here as a through membrane Up (RT to 30) safeguard, all liquidshould (high vac) have transferred in prev step Sweep all fluid to PR10.0 0.0 2.5 10.0 2.5 0.0 0.0 0.0 Continue Ramp Up (RT to 30) Aspirateliquid out of 0.0 0.0 10.0 0.0 0.0 0.0 0.0 0.0 Continue Ramp PR1 & PR2Up (RT to 30) Transfer 10 mL media 0.0 0.0 0.0 10.0 0.0 0.0 0.0 0.0Continue Ramp from Reagent Strip to Up (RT to 30) PR1 Load media fromPR1 0.0 0.0 10.0 0.5 0.0 4.5 0.0 0.0 Continue Ramp into Permeate Up (RTto 30) channel Aspirate liquid out of 0.0 0.0 0.5 0.0 4.5 0.0 0.0 0.0Continue Ramp May keep some media in PR1 & PR2 Up (RT to 30) PR1/PR2reservoirs during incubation INCUBATE SWIIN 30 C. 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 30 9 hours; may intermittently #1 - may require dealmanifold arms for intermittent airflow, airflow, media rinses mediarinses Ramp up (30 C. to 42 C.) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Ramp Up15 minutes; Ramp rate still (30 to 42) being worked by G8 INCUBATE SWIIN42 C.- 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 42 2 hours; may intermittentlymay require seal manifold arms for intermittent airflow, airflow, mediarinses media rinses Ramp down (42 C. to 30 C.) 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 Ramp Down 15 minutes; Ramp rate (42 to 30) still being worked onby G8 INCUBATE SWIIN 30 C. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 30 9 hours;may intermittently #2 - may require seal manifold arms for intermittentairflow, airflow, media rinses media rinses Pull media out of 0.0 0.00.0 0.0 0.0 10.0 0.0 0.0 Ramp Down Permeate channel (30 to RT) into PR2Aspirate liquid out of 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Continue Ramp PR2Down (30 to RT) Transfer 10 mL 0.0 0.0 0.0 10.0 0.0 0.0 0.0 0.0 ContinueRamp media + 10% glycerol Down (30 to RT) from Reagent Strip to PR1 Pullmedia + 10% glycerol 0.0 0.0 10.0 2.5 0.0 2.5 0.0 0.0 Continue Ramp fromPR1 into Down (30 to RT) Permeate channel Flood Retentate - 0.0 2.5 2.50.0 2.5 0.0 0.0 2.5 Continue Ramp Dislodge Cells Down (30 to RT) Sweepall fluid to RR2 2.5 0.0 0.0 0.0 0.0 0.0 0.0 10.0 Continue Ramp Down (30to RT) Aspirate 5 mL cell 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 Continue Rampsolution from RR2 Down (30 to RT) into final vial Aspirate liquid out of0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Continue Ramp RR & RR2 Down (30 to RT)Unload SWIIN 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Continue Ramp Down (30 toRT)

We claim:
 1. A singulation assembly for a solid wall isolation,incubation and normalization (SWIIN) module comprising: a retentatemember comprising; an upper surface and a lower surface and a first andsecond end, an upper portion of a serpentine channel defined by raisedareas on the lower surface of the retentate member, wherein the upperportion of the serpentine channel traverses the lower surface of theretentate member for about 50% to about 90% of a length and width of thelower surface of the retentate member; and at least one retentate portfluidically connected to the serpentine channel; and a permeate memberdisposed under the retentate member comprising: an upper surface and alower surface and a first and second end, a lower portion of theserpentine channel defined by raised areas on the upper surface of thepermeate member, wherein the lower portion of the serpentine channeltraverses the upper surface of the permeate member for about 50% toabout 90% of a length and width of the upper surface of the permeatemember, and wherein the lower portion of the serpentine channel isconfigured to mate with the upper portion of the serpentine channel toform the serpentine channel; and at least one permeate port fluidicallyconnected to the serpentine channel; a first gasket disposed under andadjacent to the retentate member and above and adjacent to the permeatemember; and first and second reservoirs at the first end of the permeatemember, wherein the first reservoir is fluidically connected to the atleast one permeate port in the permeate member and the second reservoiris fluidically connected to the at least one retentate port in theretentate member; a reservoir cover at the first end of the retentatemember configured to cover the first and second reservoirs; and a secondgasket disposed on top of the reservoir cover, wherein the second gasketcomprises for each of the first and second reservoirs a first and secondreservoir access aperture configured to provide fluid access to thefirst reservoir and the second reservoir, respectively, and a first andsecond pneumatic access aperture configured to provide pneumatic accessto the first reservoir and the second reservoir, respectively.
 2. TheSWIIN module of claim 1, wherein the reservoir access apertures receivefluid from outside the SWIIN module or remove fluid from the reservoirs.3. The SWIIN module of claim 1, further comprising a third and a fourthreservoir wherein the third reservoir is 1) fluidically coupled to asecond port in the retentate member, 2) fluidically coupled to a thirdreservoir access aperture in the second gasket disposed on top of thereservoir cover into which fluids and/or cells flow from outside theSWIIN module into the third reservoir, and 3) pneumatically coupled to apressure source; and wherein the fourth reservoir is 1) fluidicallycoupled to a second port in the permeate member, 2) fluidically coupledto a fourth reservoir access aperture in the second gasket on top of thereservoir cover into which fluids and/or cells flow from outside theSWIIN module into the fourth reservoir, and 3) pneumatically coupled toa pressure source.
 4. The singulation assembly of claim 1, wherein thepermeate member further comprises ultrasonic tabs disposed on the raisedareas on the upper surface of the permeate member and at the first andsecond end of the permeate member; the retentate member furthercomprises recesses for the ultrasonic tabs, wherein the recesses aredisposed on the raised areas on the lower surface of the retentatemember and at the first and second end of the retentate member; theultrasonic tabs are configured to mate with the recesses for theultrasonic tabs; and the permeate member and the retentate member arecoupled together by ultrasonic welding.
 5. The singulation assembly ofclaim 1, wherein the permeate member and the retentate member arecoupled together by a pressure sensitive adhesive.
 6. The singulationassembly of claim 1, wherein the retentate member is fabricated frompolycarbonate, cyclic olefin co-polymer, or poly(methyl methylacrylate).7. The singulation assembly of claim 1, wherein the permeate member isfabricated from polycarbonate, cyclic olefin co-polymer, or poly(methylmethylacrylate).
 8. The singulation assembly of claim 1, wherein aserpentine channel portion of each of the retentate and permeate membersis from 75 mm to 350 mm in length, from 50 mm to 250 mm in width, andfrom 2 mm to 15 mm in thickness.
 9. The singulation assembly of claim 8,wherein a serpentine channel portion of each of the retentate andpermeate members is from 150 mm to 250 mm in length, from 100 mm to 150mm in width, and from 4 mm to 8 mm in thickness.
 10. The singulationassembly of claim 1, wherein a volume of the mated serpentine channel isfrom 4 to 40 mL.
 11. The singulation assembly of claim 10, wherein thevolume of the mated serpentine channel is from 6 to 30 mL.
 12. Thesingulation assembly of claim 11, wherein the volume of the matedserpentine channel is from 10 to 20 mL.
 13. A SWIIN module comprisingthe singulation assembly of claim 1, wherein the SWIIN module furthercomprises a perforated member comprising wells positioned between theretentate member and the permeate member and imaging means to detectcells growing in the wells.
 14. The SWIIN module of claim 13, whereinthe imaging means comprises a camera and a backlight positioned beneaththe permeate member.
 15. The SWIIN module of claim 14, furthercomprising a heated cover, thermoelectric control device, and a fan. 16.A SWIIN module comprising: a singulation assembly comprising: aretentate member comprising; an upper surface and a lower surface and afirst and second end, an upper portion of a serpentine channel definedby raised areas on the lower surface of the retentate member, whereinthe upper portion of the serpentine channel traverses the lower surfaceof the retentate member for about 50% to about 90% of a length and widthof the lower surface of the retentate member; and at least one retentateport fluidically connected to the upper portion of the serpentinechannel; and a permeate member disposed under the retentate membercomprising: an upper surface and a lower surface and a first and secondend, a lower portion of the serpentine channel defined by raised areason the upper surface of the permeate member, wherein the lower portionof the serpentine channel traverses the upper surface of the permeatemember for about 50% to about 90% of a length and width of the uppersurface of the permeate member, and wherein the lower portion of theserpentine channel is configured to mate with the upper portion of theserpentine channel to form the serpentine channel; and at least onepermeate port fluidically connected to the lower portion of theserpentine channel; and a first gasket disposed under and adjacent tothe retentate member and above and adjacent to the permeate member;first and second reservoirs at the first end of the permeate member,wherein the first reservoir is fluidically connected to the at least onepermeate port in the permeate member and the second reservoir isfluidically connected to the at least one retentate port in theretentate member; a reservoir cover at the first end of the retentatemember; and a second gasket disposed on top of the reservoir cover,wherein the second gasket comprises for each of the first and secondreservoir a first and second reservoir access aperture configured toprovide fluid access to the first and second reservoirs, respectively,and a first and second pneumatic access aperture configured to providepneumatic access to the first and second reservoirs, respectively. 17.The SWIIN module of claim 16, further comprising a third and a fourthreservoir wherein the third reservoir is 1) fluidically coupled to asecond port in the retentate member, 2) fluidically coupled to a thirdreservoir access aperture in the second gasket on top of the reservoircover into which fluids and/or cells flow from outside the SWIIN moduleinto the third reservoir, and 3) pneumatically coupled to a pressuresource; and wherein the fourth reservoir is 1) fluidically coupled to asecond port in the permeate member, 2) fluidically coupled to a fourthreservoir access aperture in the second gasket on top of the reservoircover into which fluids and/or cells flow from outside the SWIIN moduleinto the fourth reservoir, and 3) pneumatically coupled to a pressuresource.
 18. The SWIIN module of claim 16, wherein the reservoir accessapertures receive fluid from outside the SWIIN module or remove fluidfrom the reservoirs.
 19. The SWIIN module of claim 17, wherein a volumeof the mated serpentine channel is from 4 to 40 mL.